Interaction Entropy: A New Paradigm for Highly Efficient and Reliable Computation of Protein-Ligand Binding Free Energy.

PubMed

Duan, Lili; Liu, Xiao; Zhang, John Z H

2016-05-04

Efficient and reliable calculation of protein-ligand binding free energy is a grand challenge in computational biology and is of critical importance in drug design and many other molecular recognition problems. The main challenge lies in the calculation of entropic contribution to protein-ligand binding or interaction systems. In this report, we present a new interaction entropy method which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein-ligand binding and other interaction processes. Drastically different from the widely employed but extremely expensive normal mode method for calculating entropy change in protein-ligand binding, the new method calculates the entropic component (interaction entropy or -TΔS) of the binding free energy directly from molecular dynamics simulation without any extra computational cost. Extensive study of over a dozen randomly selected protein-ligand binding systems demonstrated that this interaction entropy method is both computationally efficient and numerically reliable and is vastly superior to the standard normal mode approach. This interaction entropy paradigm introduces a novel and intuitive conceptual understanding of the entropic effect in protein-ligand binding and other general interaction systems as well as a practical method for highly efficient calculation of this effect.

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

PubMed

Wang, Binbin; Zhang, Huawei; Liang, Dongmei; Hao, Panlong; Li, Yanni; Qiao, Jianjun

2017-12-01

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis*

PubMed Central

León, Ileana R.; Schwämmle, Veit; Jensen, Ole N.; Sprenger, Richard R.

2013-01-01

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921

Protein requirements of bobwhite chicks for survival, growth and efficiency of feed utilization

USGS Publications Warehouse

Nestler, R.B.; Bailey, W.W.; McClure, H.E.

1942-01-01

During the summer and fall of 1939 four experiments were conducted at the Patuxent Research Refuge, Bowie, Maryland, to determine the protein requirements of bobwhite chicks. A total of 816 chicks were used to compare six levels of protein, namely, 22,24,26, 28, 30, and 32 per cent.....From the three standpoints of survival, rate of growth, and efficiency of feed utilization for the first ten weeks of life, the 28 per cent level of protein gave the best results. During the ninth and tenth weeks, the highest efficiency of feed utilization was obtained on the 22 per cent level. The results indicate that after the birds have reached about twothirds of their mature weight, the difference in efficiency between a diet containing 28 per cent of protein and one containing 22 per cent may be small enough to justify, in the interest of economy, the use of a diet containing the lower percentage of protein.

Feed efficiency of diets with different energy and protein concentrations supplemented with methionine in laying quails

NASA Astrophysics Data System (ADS)

Ratriyanto, A.; Indreswari, R.; Nuhriawangsa, A. M. P.; Purwanti, E.

2018-03-01

The study was conducted to evaluate the feed efficiency of quail diets containing different concentrations of metabolizable energy (ME) and crude protein (CP) with constant ratio and supplemented with methionine. Four hundred laying quails (Coturnix coturnix japonica) were randomly assigned to four experimental diets in a 2×2 factorial arrangement. Each dietary treatment used 5 replicates of 20 quails. Two basal diets were formulated to contain 2,800 kcal kg-1 ME and 18.7% CP (High ME-CP) and 2,600 kcal kg-1 ME and 17.3% CP (Low ME-CP). Each basal diet was supplemented with 0 and 0.12% methionine. The High ME-CP diets generated lower feed consumption but higher egg mass and feed efficiency (P<0.01) compared with the Low ME-CP. Furthermore, supplementation of methionine increased egg mass, feed efficiency, energy efficiency ratio and protein efficiency ratio (P<0.01). The High ME-CP supplemented with methionine resulted the highest feed efficiency followed by the Low ME-CP supplemented with methionine, while both High ME-CP and Low ME-CP without methionine supplementation resulted the lowest feed efficiency (P<0.05). In addition, ME and CP consumption of the birds were not influenced by the treatments. Thus, feeding High ME-CP supplemented with 0.12% methionine provided benefit to improve the feed efficiency in laying quails.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

2008-08-01

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Responsive Proteins in Wheat Cultivars with Contrasting Nitrogen Efficiencies under the Combined Stress of High Temperature and Low Nitrogen

PubMed Central

Abd_Allah, Elsayed Fathi; Nauman, Mohd; Asif, Ambreen; Hashem, Abeer; Alqarawi, Abdulaziz A.

2017-01-01

Productivity of wheat (Triticum aestivum) is markedly affected by high temperature and nitrogen deficiency. Identifying the functional proteins produced in response to these multiple stresses acting in a coordinated manner can help in developing tolerance in the crop. In this study, two wheat cultivars with contrasting nitrogen efficiencies (N-efficient VL616 and N-inefficient UP2382) were grown in control conditions, and under a combined stress of high temperature (32 °C) and low nitrogen (4 mM), and their leaf proteins were analysed in order to identify the responsive proteins. Two-dimensional electrophoresis unravelled sixty-one proteins, which varied in their expression in wheat, and were homologous to known functional proteins involved in biosynthesis, carbohydrate metabolism, energy metabolism, photosynthesis, protein folding, transcription, signalling, oxidative stress, water stress, lipid metabolism, heat stress tolerance, nitrogen metabolism, and protein synthesis. When exposed to high temperature in combination with low nitrogen, wheat plants altered their protein expression as an adaptive means to maintain growth. This response varied with cultivars. Nitrogen-efficient cultivars showed a higher potential of redox homeostasis, protein stability, osmoprotection, and regulation of nitrogen levels. The identified stress-responsive proteins can pave the way for enhancing the multiple-stress tolerance in wheat and developing a better understanding of its mechanism. PMID:29186028

Effect of increasing dietary metabolizable protein on nitrogen efficiency in Holstein dairy cows

PubMed Central

Imran, Muhammad; Pasha, Talat Naseer; Shahid, Muhammad Qamer; Babar, Imran; Naveed ul Haque, Muhammad

2017-01-01

Objective The objective of the study was to determine the effects of increasing levels of metabolizable protein (MP) on lactation performance and nitrogen (N) efficiencies in lactating dairy cows. Methods Nine multiparous cows in mid lactation [113±25 days in milk] received three treatments in a 3×3 Latin square design with a period length of 21 days. The treatments were three diets, designed to provide similar energy and increasing supply of MP (g/d) (2,371 [low], 2,561 [medium], and 2,711 [high] with corresponding crude protein levels [%]) 15.2, 18.4, and 20.9, respectively. Results Increasing MP supplies did not modify dry matter intake, however, it increased milk protein, fat, and lactose yield linearly. Similarly, fat corrected milk increased linearly (9.3%) due to an increase in both milk yield (5.2%) and milk fat content (7.8%). No effects were observed on milk protein and lactose contents across the treatments. Milk nitrogen efficiency (MNE) decreased from 0.26 to 0.20; whereas, the metabolic efficiency of MP decreased from 0.70 to 0.60 in low to high MP supplies, respectively. The concentration of blood urea nitrogen (BUN) increased linearly in response to increasing MP supplies. Conclusion Increasing MP supplies resulted in increased milk protein yield; however, a higher BUN and low MNE indicated an efficient utilization of dietary protein at low MP supplies. PMID:28002937

High efficiency protein separation with organosilane assembled silica coated magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Chang, Jeong Ho; Kang, Ki Ho; Choi, Jinsub; Jeong, Young Keun

2008-10-01

This work describes the development of high efficiency protein separation with functionalized organosilanes on the surface of silica coated magnetic nanoparticles. The magnetic nanoparticles were synthesized with average particle size of 9 nm and silica coated magnetic nanoparticles were obtained by controlling the coating thicknesses on magnetic nanoparticles. The silica coating thickness could be uniformly sized with a diameter of 10-40 nm by a sol-gel approach. The surface modification was performed with four kinds of functionalized organosilanes such as carboxyl, aldehyde, amine, and thiol groups. The protein separation work with organosilane assembled silica coated magnetic nanoparticles was achieved for model proteins such as bovine serum albumin (BSA) and lysozyme (LSZ) at different pH conditions. Among the various functionalities, the thiol group showed good separation efficiency due to the change of electrostatic interactions and protein conformational structure. The adsorption efficiency of BSA and LSZ was up to 74% and 90% corresponding pH 4.65 and pH 11.

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo

2008-12-05

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminalmore » of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 {mu}M, and appeared to plateau above a concentration of 1 {mu}M. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.« less

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

PubMed

Wang, Ming; Zuris, John A; Meng, Fantao; Rees, Holly; Sun, Shuo; Deng, Pu; Han, Yong; Gao, Xue; Pouli, Dimitra; Wu, Qi; Georgakoudi, Irene; Liu, David R; Xu, Qiaobing

2016-03-15

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Feed conversion efficiency in aquaculture: do we measure it correctly?

NASA Astrophysics Data System (ADS)

Fry, Jillian P.; Mailloux, Nicholas A.; Love, David C.; Milli, Michael C.; Cao, Ling

2018-02-01

Globally, demand for food animal products is rising. At the same time, we face mounting, related pressures including limited natural resources, negative environmental externalities, climate disruption, and population growth. Governments and other stakeholders are seeking strategies to boost food production efficiency and food system resiliency, and aquaculture (farmed seafood) is commonly viewed as having a major role in improving global food security based on longstanding measures of animal production efficiency. The most widely used measurement is called the ‘feed conversion ratio’ (FCR), which is the weight of feed administered over the lifetime of an animal divided by weight gained. By this measure, fed aquaculture and chickens are similarly efficient at converting feed into animal biomass, and both are more efficient compared to pigs and cattle. FCR does not account for differences in feed content, edible portion of an animal, or nutritional quality of the final product. Given these limitations, we searched the literature for alternative efficiency measures and identified ‘nutrient retention’, which can be used to compare protein and calories in feed (inputs) and edible portions of animals (outputs). Protein and calorie retention have not been calculated for most aquaculture species. Focusing on commercial production, we collected data on feed composition, feed conversion ratios, edible portions (i.e. yield), and nutritional content of edible flesh for nine aquatic and three terrestrial farmed animal species. We estimate that 19% of protein and 10% of calories in feed for aquatic species are ultimately made available in the human food supply, with significant variation between species. Comparing all terrestrial and aquatic animals in the study, chickens are most efficient using these measures, followed by Atlantic salmon. Despite lower FCRs in aquaculture, protein and calorie retention for aquaculture production is comparable to livestock production. This is, in part, due to farmed fish and shrimp requiring higher levels of protein and calories in feed compared to chickens, pigs, and cattle. Strategies to address global food security should consider these alternative efficiency measures.

Reconstruction of calmodulin single-molecule FRET states, dye interactions, and CaMKII peptide binding by MultiNest and classic maximum entropy

NASA Astrophysics Data System (ADS)

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-08-01

We analyzed single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy

PubMed Central

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-01-01

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data. PMID:24223465

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy.

PubMed

Devore, Matthew S; Gull, Stephen F; Johnson, Carey K

2013-08-30

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca 2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

PubMed Central

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface. PMID:29360877

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

PubMed

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens.

PubMed

Toyomizu, Masaaki; Kikusato, Motoi; Kawabata, Yusuke; Azad, Md Abul Kalam; Inui, Eriko; Amo, Taku

2011-05-01

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens. Copyright © 2011 Elsevier Inc. All rights reserved.

Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

PubMed Central

Suomalainen, M; Garoff, H

1994-01-01

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways. Images PMID:8035486

Efficient marginalization to compute protein posterior probabilities from shotgun mass spectrometry data

PubMed Central

Serang, Oliver; MacCoss, Michael J.; Noble, William Stafford

2010-01-01

The problem of identifying proteins from a shotgun proteomics experiment has not been definitively solved. Identifying the proteins in a sample requires ranking them, ideally with interpretable scores. In particular, “degenerate” peptides, which map to multiple proteins, have made such a ranking difficult to compute. The problem of computing posterior probabilities for the proteins, which can be interpreted as confidence in a protein’s presence, has been especially daunting. Previous approaches have either ignored the peptide degeneracy problem completely, addressed it by computing a heuristic set of proteins or heuristic posterior probabilities, or by estimating the posterior probabilities with sampling methods. We present a probabilistic model for protein identification in tandem mass spectrometry that recognizes peptide degeneracy. We then introduce graph-transforming algorithms that facilitate efficient computation of protein probabilities, even for large data sets. We evaluate our identification procedure on five different well-characterized data sets and demonstrate our ability to efficiently compute high-quality protein posteriors. PMID:20712337

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

Development of an inducible platform for intercellular protein delivery.

PubMed

Siller, Richard; Dufour, Eric; Lycke, Max; Wilmut, Ian; Jung, Yong-Wook; Park, In Hyun; Sullivan, Gareth J

2017-04-30

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

An analysis of partial efficiencies of energy utilisation of different macronutrients by barramundi (Lates calcarifer) shows that starch restricts protein utilisation in carnivorous fish.

PubMed

Glencross, Brett D; Blyth, David; Bourne, Nicholas; Cheers, Susan; Irvin, Simon; Wade, Nicholas M

2017-02-01

This study examined the effect of including different dietary proportions of starch, protein and lipid, in diets balanced for digestible energy, on the utilisation efficiencies of dietary energy by barramundi (Lates calcarifer). Each diet was fed at one of three ration levels (satiety, 80 % of initial satiety and 60 % of initial satiety) for a 42-d period. Fish performance measures (weight gain, feed intake and feed conversion ratio) were all affected by dietary energy source. The efficiency of energy utilisation was significantly reduced in fish fed the starch diet relative to the other diets, but there were no significant effects between the other macronutrients. This reduction in efficiency of utilisation was derived from a multifactorial change in both protein and lipid utilisation. The rate of protein utilisation deteriorated as the amount of starch included in the diet increased. Lipid utilisation was most dramatically affected by inclusion levels of lipid in the diet, with diets low in lipid producing component lipid utilisation rates well above 1·3, which indicates substantial lipid synthesis from other energy sources. However, the energetic cost of lipid gain was as low as 0·65 kJ per kJ of lipid deposited, indicating that barramundi very efficiently store energy in the form of lipid, particularly from dietary starch energy. This study defines how the utilisation efficiency of dietary digestible energy by barramundi is influenced by the macronutrient source providing that energy, and that the inclusion of starch causes problems with protein utilisation in this species.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

PubMed

Pham, Ha H; Szent-Gyorgyi, Christopher; Brotherton, Wendy L; Schmidt, Brigitte F; Zanotti, Kimberly J; Waggoner, Alan S; Armitage, Bruce A

2015-03-28

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Bichromophoric Dyes for Wavelength Shifting of Dye-Protein Fluoromodules

PubMed Central

Pham, Ha H.; Szent-Gyorgyi, Christopher; Brotherton, Wendy L.; Schmidt, Brigitte F.; Zanotti, Kimberly J.; Waggoner, Alan S.

2015-01-01

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields. PMID:25679477

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

PubMed Central

Rodriguez, Erik A.; Lester, Henry A.; Dougherty, Dennis A.

2007-01-01

The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased ΔG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS′, which shows ∼50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible. PMID:17698637

Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

PubMed

Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

2016-01-01

Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds

PubMed Central

2011-01-01

Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product. PMID:21548923

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

PubMed Central

Lakkaraju, Asvin K. K.; Thankappan, Ratheeshkumar; Mary, Camille; Garrison, Jennifer L.; Taunton, Jack; Strub, Katharina

2012-01-01

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120–160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway. PMID:22648169

Efficient Multicriteria Protein Structure Comparison on Modern Processor Architectures

PubMed Central

Manolakos, Elias S.

2015-01-01

Fast increasing computational demand for all-to-all protein structures comparison (PSC) is a result of three confounding factors: rapidly expanding structural proteomics databases, high computational complexity of pairwise protein comparison algorithms, and the trend in the domain towards using multiple criteria for protein structures comparison (MCPSC) and combining results. We have developed a software framework that exploits many-core and multicore CPUs to implement efficient parallel MCPSC in modern processors based on three popular PSC methods, namely, TMalign, CE, and USM. We evaluate and compare the performance and efficiency of the two parallel MCPSC implementations using Intel's experimental many-core Single-Chip Cloud Computer (SCC) as well as Intel's Core i7 multicore processor. We show that the 48-core SCC is more efficient than the latest generation Core i7, achieving a speedup factor of 42 (efficiency of 0.9), making many-core processors an exciting emerging technology for large-scale structural proteomics. We compare and contrast the performance of the two processors on several datasets and also show that MCPSC outperforms its component methods in grouping related domains, achieving a high F-measure of 0.91 on the benchmark CK34 dataset. The software implementation for protein structure comparison using the three methods and combined MCPSC, along with the developed underlying rckskel algorithmic skeletons library, is available via GitHub. PMID:26605332

Structure and properties of hybrid biopolymer particles fabricated by co-precipitation cross-linking dissolution procedure.

PubMed

Xiong, Yu; Georgieva, Radostina; Steffen, Axel; Smuda, Kathrin; Bäumler, Hans

2018-03-15

The Co-precipitation Crosslinking Dissolution technique (CCD-technique) allows a few-steps fabrication of particles composed of different biopolymers and bioactive agents under mild conditions. Morphology and properties of the fabricated biopolymer particles depend on the fabrication conditions, the nature of the biopolymers and additives, but also on the choice of the inorganic templates for co-precipitation. Here, we investigate the influence of an acidic biopolymer, hyaluronic acid (HA), on the formation of particles from bovine hemoglobin and bovine serum albumin applying co-precipitation with CaCO 3 and MnCO 3 . CaCO 3 templated biopolymer particles are almost spherical with particle size from 2 to 20 µm and protein entrapment efficiency from 13 to 77%. Presence of HA causes significant structural changes of the particles and decreasing protein entrapment efficiency. In contrast, MnCO 3 templated particles exhibit uniform peanut shape and submicron size with remarkably high protein entrapment efficiency of nearly 100%. Addition of HA has no influence on the protein entrapment efficiency or on morphology and size of the particles. These effects can be attributed to the strong interaction of Mn 2+ with proteins and much weaker interaction with HA. Therefore, entrapment efficiency, size and structure of biopolymer particles can be optimized by varying the mineral templates and additives. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient Multicriteria Protein Structure Comparison on Modern Processor Architectures.

PubMed

Sharma, Anuj; Manolakos, Elias S

2015-01-01

Fast increasing computational demand for all-to-all protein structures comparison (PSC) is a result of three confounding factors: rapidly expanding structural proteomics databases, high computational complexity of pairwise protein comparison algorithms, and the trend in the domain towards using multiple criteria for protein structures comparison (MCPSC) and combining results. We have developed a software framework that exploits many-core and multicore CPUs to implement efficient parallel MCPSC in modern processors based on three popular PSC methods, namely, TMalign, CE, and USM. We evaluate and compare the performance and efficiency of the two parallel MCPSC implementations using Intel's experimental many-core Single-Chip Cloud Computer (SCC) as well as Intel's Core i7 multicore processor. We show that the 48-core SCC is more efficient than the latest generation Core i7, achieving a speedup factor of 42 (efficiency of 0.9), making many-core processors an exciting emerging technology for large-scale structural proteomics. We compare and contrast the performance of the two processors on several datasets and also show that MCPSC outperforms its component methods in grouping related domains, achieving a high F-measure of 0.91 on the benchmark CK34 dataset. The software implementation for protein structure comparison using the three methods and combined MCPSC, along with the developed underlying rckskel algorithmic skeletons library, is available via GitHub.

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

PubMed

Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mie, Masayasu; Shimizu, Shun; Takahashi, Fumio

2008-08-15

The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, onlymore » mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library.« less

Energy consumption and water-soluble protein release by cell wall disruption of Nannochloropsis gaditana.

PubMed

Safi, C; Cabas Rodriguez, L; Mulder, W J; Engelen-Smit, N; Spekking, W; van den Broek, L A M; Olivieri, G; Sijtsma, L

2017-09-01

Several cell disruption methods were tested on Nannochloropsis gaditana, to evaluate their efficiency in terms of cell disintegration, energy input and release of soluble proteins. High-pressure homogenization (HPH) and bead milling were the most efficient with >95% cell disintegration, ±50% (w/w) release of total proteins and low energy input (<0.5kWh.kg -1 biomass ). Enzymatic treatment required low energy input (<0.34kWh.kg -1 biomass ), but it only released ±35% protein (w/w). Pulsed Electric Field (PEF) was neither energy-efficient (10.44kWh.kg -1 biomass ) nor successful for protein release (only 10% proteins w/w) and cell disintegration. The release of proteins after applying HPH and bead milling always required less intensive operating conditions for cell disruption. The energy cost per unit of released protein ranged from 0.15-0.25 €.kg Protein -1 in case of HPH, and up to 2-20 €.kg Protein -1 in case of PEF. Copyright © 2017 Elsevier Ltd. All rights reserved.

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential.

PubMed

Brgles, Marija; Jurasin, Darija; Sikirić, Maja Dutour; Frkanec, Ruza; Tomasić, Jelka

2008-01-01

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.

A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis.

PubMed

Keith, D; Hong, B; Christensen, M

1997-05-01

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography

PubMed Central

Ito, Yoichiro; Clary, Robert

2016-01-01

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1–2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate. PMID:27790621

Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography.

PubMed

Ito, Yoichiro; Clary, Robert

2016-12-01

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1-2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate.

Detection of links between Ebola nucleocapsid and virulence using disorder analysis.

PubMed

Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

2015-08-01

The underlying reasons for the differences in the virulence of various types of Ebola virus (EBOV) remain unknown. Comparison of the percentage of disorder (PID) in nucleocapsid proteins VP30 and NP reveals high correlation between nucleocapsid PIDs and the case-fatality rates of EBOV. The higher disorder of these proteins is likely to be needed for more efficient multiplication of virus copies via more efficient viral RNA transcription and more promiscuous protein binding potential. This is important for the more efficient assistance of nucleocapsid in viral particle budding and of the assembly and mobility of viral proteins across the host membrane and within the cytoplasm. A more comprehensive knowledge of the molecular mechanisms of EBOV virulence would also lead to new and more effective strategies in vaccine development.

Protein immobilization onto various surfaces using a polymer-bound isocyanate

NASA Astrophysics Data System (ADS)

Kang, Hyun-Jin; Cha, Eun Ji; Park, Hee-Deung

2015-01-01

Silane coupling agents have been widely used for immobilizing proteins onto inorganic surfaces. However, the immobilization method using silane coupling agents requires several treatment steps, and its application is limited to only surfaces containing hydroxyl groups. The aim of this study was to develop a novel method to overcome the limitations of the silane-based immobilization method using a polymer-bound isocyanate. Initially, polymer-bound isocyanate was dissolved in organic solvent and then was used to dip-coat inorganic surfaces. Proteins were then immobilized onto the dip-coated surfaces by the formation of urea bonds between the isocyanate groups of the polymer and the amine groups of the protein. The reaction was verified by FT-IR in which NCO stretching peaks disappeared, and CO and NH stretching peaks appeared after immobilization. The immobilization efficiency of the newly developed method was insensitive to reaction temperatures (4-50 °C), but the efficiency increased with reaction time and reached a maximum after 4 h. Furthermore, the method showed comparable immobilization efficiency to the silane-based immobilization method and was applicable to surfaces that cannot form hydroxyl groups. Taken together, the newly developed method provides a simple and efficient platform for immobilizing proteins onto surfaces.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

PubMed

Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian

2014-12-01

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

In silico analysis of different generation β lactams antibiotics with penicillin binding protein-2 of Neisseria meningitidis for curing meningococcal disease.

PubMed

Tripathi, Vijay; Tripathi, Pooja; Srivastava, Navita; Gupta, Dwijendra

2014-12-01

Neisseria meningitidis is a gram negative, diplococcic pathogen responsible for the meningococcal disease and fulminant septicemia. Penicillin-binding proteins-2 (PBPs) is crucial for the cell wall biosynthesis during cell proliferation of N. meningitidis and these are the target for β-lactam antibiotics. For many years penicillin has been recognized as the antibiotic for meningococcal disease but the meningococcus has seemed to be antibiotic resistance. In the present work we have verified the molecular interaction of Penicillin binding protein-2 N. meningitidis to different generation of β-lactam antibiotics and concluded that the third generation of β-lactam antibiotics shows efficient binding with Penicillin binding protein-2 of N. meningitidis. On the basis of binding efficiency and inhibition constant, ceftazidime emerged as the most efficient antibiotic amongst the other advanced β-lactam antibiotics against Penicillin-binding protein-2 of N. meningitidis.

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Thermal sensitivity of mitochondrial respiration efficiency and protein phosphorylation in the clam Mercenaria mercenaria.

PubMed

Ulrich, P N; Marsh, A G

2009-01-01

The mitochondria of intertidal invertebrates continue to function when organisms are exposed to rapid substantial shifts in temperature. To test if mitochondrial physiology of the clam Mercenaria mercenaria is compromised under elevated temperatures, we measured mitochondrial respiration efficiency at 15 degrees C, 18 degrees C, and 21 degrees C using a novel, high-throughput, microplate respirometry methodology developed for this study. Though phosphorylating (state 3) and resting (state 4) respiration rates were unaffected over this temperature range, respiratory control ratios (RCRs: ratio of state 3 to state 4 respiration rates) decreased significantly above 18 degrees C (p < 0.05). The drop in RCR was not associated with reduction of phosphorylation efficiency, suggesting that, while aerobic scope of mitochondrial respiration is limited at elevated temperatures, mitochondria continue to efficiently produce adenosine triphosphate. We further investigated the response of clam mitochondria to elevated temperatures by monitoring phosphorylation of mitochondrial protein. Three proteins clearly demonstrated significant time- and temperature-specific phosphorylation patterns. The protein-specific patterns of phosphorylation may suggest that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature. Thus, while aerobic scope of clam mitochondrial respiration is reduced at moderate temperatures, specific protein phosphorylation responses reflect large shifts in function that are initiated within the organelle at higher temperatures.

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

PubMed Central

Xia, Xuhua

2015-01-01

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation ITE is formulated to facilitate studies on the efficiency and evolution of the translation machinery. PMID:25480780

Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

PubMed

de Jesus, Jemmyson Romário; da Silva Fernandes, Rafael; de Souza Pessôa, Gustavo; Raimundo, Ivo Milton; Arruda, Marco Aurélio Zezzi

2017-08-01

The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL -1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL -1 . Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

Carnot cycle at finite power: attainability of maximal efficiency.

PubMed

Allahverdyan, Armen E; Hovhannisyan, Karen V; Melkikh, Alexey V; Gevorkian, Sasun G

2013-08-02

We want to understand whether and to what extent the maximal (Carnot) efficiency for heat engines can be reached at a finite power. To this end we generalize the Carnot cycle so that it is not restricted to slow processes. We show that for realistic (i.e., not purposefully designed) engine-bath interactions, the work-optimal engine performing the generalized cycle close to the maximal efficiency has a long cycle time and hence vanishing power. This aspect is shown to relate to the theory of computational complexity. A physical manifestation of the same effect is Levinthal's paradox in the protein folding problem. The resolution of this paradox for realistic proteins allows to construct engines that can extract at a finite power 40% of the maximally possible work reaching 90% of the maximal efficiency. For purposefully designed engine-bath interactions, the Carnot efficiency is achievable at a large power.

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

PubMed

Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-08-07

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

Macrofaunal involvement in the sublittoral decay of kelp debris: The sea urchin Psammechinus miliaris (Gmelin) (Echinodermata: Echinoidea)

NASA Astrophysics Data System (ADS)

Bedford, A. P.; Moore, P. G.

1985-01-01

Psammechinus miliaris occurs in the Clyde Sea area in large numbers (<18 individuals per 100 g -1 weed dry wt) on sublittoral beds of detached Laminaria saccharina. Its rôle in weed decomposition has been examined by comparing its responses (behavioural choice, growth rate, absorption efficiencies of both carbon and protein, gut retention times and rate of faecal output) to fresh and rotting weed. Younger urchins grew faster than older individuals on a diet of rotting weed but not on fresh weed. Large seasonal variation existed, however, with fast growth occurring in June-August and little, or no, growth in December-February, irrespective of diet. Starved controls did not grow. Correcting for seasonality, rotting kelp still promoted faster growth of young urchins than did fresh weed. Larger (older) individuals showed no difference. Urchins fed fresh weed had significantly longer gut retention times. Protein absorption efficiency was higher on fresh than rotting weed, varying with weed protein content and size of urchin. Very young individuals can only digest high protein weed efficiently, eg. material derived from near the frond meristem. Organic carbon content of rotting weed was significantly lower than fresh weed. Carbon absorption efficiencies were significantly higher on fresh weed which related to organic carbon content. Standard-sized urchins fed rotting weed produced larger dry weights of faeces per day, reflecting increased ingestion rate. In closed-system choice experiments urchins preferred rotting weed kinetically. Size-frequency analysis of field populations suggested that weed beds are principally colonized by larval settlement from the plankton. Mature Psammechinus have evolved different 'strategies' for exploiting fresh and rotting weed. Fresh weed is relatively difficult to digest and long gut retention times allow high protein absorption efficiencies to be attained. Rotting weed has microbial protein in quantities and a lower organic carbon fraction. Some bacterial protein is seemingly unavailable though and lower protein absorption efficiencies result. Thus gut retention time is shortened and more food passed through the gut. Growth remains equivalent. Substratum digestion is of paramount importance for Psammechinus feeding on either fresh or rotting weed, cf. the 'classical' microbe-stripping detritivore of Fenchel.

Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.

PubMed

Hoang, Margaret L; Leon, Ronald P; Pessoa-Brandao, Luis; Hunt, Sonia; Raghuraman, M K; Fangman, Walton L; Brewer, Bonita J; Sclafani, Robert A

2007-11-01

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

BiP clustering facilitates protein folding in the endoplasmic reticulum.

PubMed

Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

2014-07-01

The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Mutual physiological genetic mechanism of plant high water use efficiency and nutrition use efficiency.

PubMed

Cao, Hong-Xing; Zhang, Zheng-Bin; Xu, Ping; Chu, Li-Ye; Shao, Hong-Bo; Lu, Zhao-Hua; Liu, Jun-Hong

2007-05-15

Water deficiency and lower fertilizer utilization efficiency are major constraints of productivity and yield stability. Improvements of crop water use efficiency (WUE) and nutrient use efficiency (NUE) is becoming an important objective in crop breeding. With the introduction of new physiological and biological approaches, we can better understand the mutual genetics mechanism of high use efficiency of water and nutrient. Much work has been done in past decades mainly including the interactions between different fertilizers and water influences on root characteristics and crop growth. Fertilizer quantity and form were regulated in order to improve crop WUE. The crop WUE and NUE shared the same increment tendency during evolution process; some genes associated with WUE and NUE have been precisely located and marked on the same chromosomes, some genes related to WUE and NUE have been cloned and transferred into wheat and rice and other plants, they can enhance water and nutrient use efficiency. The proteins transporting nutrient and water were identified such as some water channel proteins. The advance on the mechanism of higher water and nutrient use efficiency in crop was reviewed in this article, and it could provide some useful information for further research on WUE and NUE in crop.

Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro.

PubMed

Yao, Weijing; Zha, Qian; Cheng, Xu; Wang, Xin; Wang, Jun; Tang, Rupei

2016-11-23

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids. © The Author(s) 2016.

Maximizing recovery of water-soluble proteins through acetone precipitation.

PubMed

Crowell, Andrew M J; Wall, Mark J; Doucette, Alan A

2013-09-24

Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation. In 80% acetone, the precipitation efficiency for six of 10 protein standards was poor (ca. ≤15%). Poor recovery was also observed for proteome extracts, including bacterial and mammalian cells. As shown in this work, increasing the ionic strength of the solution dramatically improves the precipitation efficiency of individual proteins, and proteome mixtures (ca. 80-100% yield). This is obtained by including 1-30 mM NaCl, together with acetone (50-80%) which maximizes protein precipitation efficiency. The amount of salt required to restore the recovery correlates with the amount of protein in the sample, as well as the intrinsic protein charge, and the dielectric strength of the solution. This synergistic approach to protein precipitation in acetone with salt is consistent with a model of ion pairing in organic solvent, and establishes an improved method to recover proteins and proteome mixtures in high yield. Copyright © 2013 Elsevier B.V. All rights reserved.

Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

PubMed Central

Zhang, Yan; Zhang, Ting; Feng, Yanye; Lu, Xiuxiu; Lan, Wenxian; Wang, Jufang; Wu, Houming; Cao, Chunyang; Wang, Xiaoning

2011-01-01

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes. PMID:21829569

Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro, E-mail: fujii@sapmed.ac.j

2011-05-25

Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression ofmore » C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.« less

Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.

PubMed

Fallahbaghery, Azadeh; Zou, Wei; Byrne, Keren; Howitt, Crispin A; Colgrave, Michelle L

2017-04-05

The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.

Preparation of non-porous microspheres with high entrapment efficiency of proteins by a (water-in-oil)-in-oil emulsion technique.

PubMed

Viswanathan, N B; Thomas, P A; Pandit, J K; Kulkarni, M G; Mashelkar, R A

1999-03-08

Emulsification-solvent removal methods have been widely used for encapsulating bioactive macromolecules like proteins and polypeptides in biodegradable polymers. We report, a (water-in-oil)-in-oil emulsion technique wherein proteins and polypeptides differing in molecular weight and shape were encapsulated in polymers of current biomedical interest. When an oil was used as the processing medium in combination with a carefully selected mixed solvent system such that a stable (w/o1/o2 emulsion is formed and solvents are removed by a combination of extraction and evaporation, the entrapment efficiency was high and the product nonporous. The entrapment efficiency of globular proteins exceeded 90% while that of fibrous proteins was around 70%. Fracture studies revealed that the polymer matrix was dense. The mechanism of entrapment involved solvent-induced precipitation of the protein as the microspheres were being formed. The principle of the method will find use in preparation of non-porous polymer microparticles with reduced burst effect.

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

PubMed

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein import into isolated pea root leucoplasts.

PubMed

Chu, Chiung-Chih; Li, Hsou-Min

2015-01-01

Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that prefer Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

Highly Efficient Computation of the Basal kon using Direct Simulation of Protein-Protein Association with Flexible Molecular Models.

PubMed

Saglam, Ali S; Chong, Lillian T

2016-01-14

An essential baseline for determining the extent to which electrostatic interactions enhance the kinetics of protein-protein association is the "basal" kon, which is the rate constant for association in the absence of electrostatic interactions. However, since such association events are beyond the milliseconds time scale, it has not been practical to compute the basal kon by directly simulating the association with flexible models. Here, we computed the basal kon for barnase and barstar, two of the most rapidly associating proteins, using highly efficient, flexible molecular simulations. These simulations involved (a) pseudoatomic protein models that reproduce the molecular shapes, electrostatic, and diffusion properties of all-atom models, and (b) application of the weighted ensemble path sampling strategy, which enhanced the efficiency of generating association events by >130-fold. We also examined the extent to which the computed basal kon is affected by inclusion of intermolecular hydrodynamic interactions in the simulations.

Modifications in trypsin digestion protocol for increasing the efficiency and coverage.

PubMed

Syal, Kirtimaan; Tadala, Raghu

2015-01-01

Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of 'staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2.

PubMed

Suetsugu, S; Miki, H; Yamaguchi, H; Obinata, T; Takenawa, T

2001-12-01

The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

PubMed Central

2012-01-01

Background Whole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells. Results The highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts. Conclusions BTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors. PMID:22559081

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.

PubMed

Brockmann, Eeva-Christine; Lamminmäki, Urpo; Saviranta, Petri

2005-06-20

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.

Efficient genome editing by FACS enrichment of paired D10A Cas9 nickases coupled with fluorescent proteins.

PubMed

Gopalappa, Ramu; Song, Myungjae; Chandrasekaran, Arun Pandian; Das, Soumyadip; Haq, Saba; Koh, Hyun Chul; Ramakrishna, Suresh

2018-05-31

Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of double-nicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.

Speed over efficiency: locusts select body temperatures that favour growth rate over efficient nutrient utilization

PubMed Central

Miller, Gabriel A.; Clissold, Fiona J.; Mayntz, David; Simpson, Stephen J.

2009-01-01

Ectotherms have evolved preferences for particular body temperatures, but the nutritional and life-history consequences of such temperature preferences are not well understood. We measured thermal preferences in Locusta migratoria (migratory locusts) and used a multi-factorial experimental design to investigate relationships between growth/development and macronutrient utilization (conversion of ingesta to body mass) as a function of temperature. A range of macronutrient intake values for insects at 26, 32 and 38°C was achieved by offering individuals high-protein diets, high-carbohydrate diets or a choice between both. Locusts placed in a thermal gradient selected temperatures near 38°C, maximizing rates of weight gain; however, this enhanced growth rate came at the cost of poor protein and carbohydrate utilization. Protein and carbohydrate were equally digested across temperature treatments, but once digested both macronutrients were converted to growth most efficiently at the intermediate temperature (32°C). Body temperature preference thus yielded maximal growth rates at the expense of efficient nutrient utilization. PMID:19625322

A highly efficient approach to protein interactome mapping based on collaborative filtering framework.

PubMed

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-09

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly.

A Highly Efficient Approach to Protein Interactome Mapping Based on Collaborative Filtering Framework

PubMed Central

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-01

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly. PMID:25572661

A Highly Efficient Approach to Protein Interactome Mapping Based on Collaborative Filtering Framework

NASA Astrophysics Data System (ADS)

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-01

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly.

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

PubMed

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly efficient antibody immobilization with multimeric protein Gs coupled magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Lee, J. H.; Choi, H. K.; Chang, J. H.

2011-10-01

This work reports the immobilization of monomeric, dimeric and trimer protein Gs onto silica magnetic nanoparticles for self-oriented antibody immobilization. To achieve this, we initially prepared the silica-coated magnetic nanoparticle having about 170 nm diameters. The surface of the silica coated magnetic nanoparticles was modified with 3- aminopropyl-trimethoxysilane (APTMS) to chemically link to multimeric protein Gs. The conjugation of amino groups on the SiO2-MNPs to cysteine tagged in multimeric protein Gs was performed using a sulfo-SMCC coupling procedure. The binding efficiencies of monomer, dimer and trimer were 77 %, 67 % and 55 % respectively. However, the efficiencies of antibody immobilization were 70 %, 83 % and 95 % for monomeric, dimeric and trimeric protein G, respectively. To prove the enhancement of accessibility by using multimeric protein G, FITC labeled goat-anti-mouse IgG was treated to mouse IgG immobilized magnetic silica nanoparticles through multimeric protein G. FITC labeled goat anti-mouse IgGs were more easily bound to mouse IgG immobilized by trimeric protein G than others. Finally protein G bound silica magnetic nanoparticles were utilized to develop highly sensitive immunoassay to detect hepatitis B antigen.

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

Functional Characterisation of the WW Minimal Domain for Delivering Therapeutic Proteins by Adenovirus Dodecahedron

PubMed Central

Villegas-Méndez, Ana; Fender, Pascal; Garin, Marina I.; Rothe, Romy; Liguori, Lavinia; Marques, Bruno; Lenormand, Jean-Luc

2012-01-01

Protein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron) has recently been shown to deliver proteins fused to the tandem WW2-3-4 structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW3 and its mutant form WW3_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application. PMID:23028993

Relationship of nitrogen use efficiency with the activities of enzymes involved in nitrogen uptake and assimilation of finger millet genotypes grown under different nitrogen inputs.

PubMed

Gupta, Nidhi; Gupta, Atul K; Gaur, Vikram S; Kumar, Anil

2012-01-01

Nitrogen responsiveness of three-finger millet genotypes (differing in their seed coat colour) PRM-1 (brown), PRM-701 (golden), and PRM-801 (white) grown under different nitrogen doses was determined by analyzing the growth, yield parameters and activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase; GOGAT, and glutamate dehydrogenase (GDH) at different developmental stages. High nitrogen use efficiency and nitrogen utilization efficiency were observed in PRM-1 genotype, whereas high nitrogen uptake efficiency was observed in PRM-801 genotype. At grain filling nitrogen uptake efficiency in PRM-1 negatively correlated with NR, GS, GOGAT activities whereas it was positively correlated in PRM-701 and PRM-801, however, GDH showed a negative correlation. Growth and yield parameters indicated that PRM-1 responds well at high nitrogen conditions while PRM-701 and PRM-801 respond well at normal and low nitrogen conditions respectively. The study indicates that PRM-1 is high nitrogen responsive and has high nitrogen use efficiency, whereas golden PRM-701 and white PRM-801 are low nitrogen responsive genotypes and have low nitrogen use efficiency. However, the crude grain protein content was higher in PRM-801 genotype followed by PRM-701 and PRM-1, indicating negative correlation of nitrogen use efficiency with source to sink relationship in terms of seed protein content.

Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

PubMed Central

Zhu, Jinge; Rao, Hongyu; Tonelli, Marco; Westler, Milo; Singarapu, Kiran K.; Markley, John L.; DeLuca, Hector F.; Assadi-Porter, Fariba M.

2012-01-01

Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from E. coli inclusion bodies. The heterologously expressed protein constructs retain full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with, 13C, and 15N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed. PMID:22750673

Chromophore Isomer Stabilization Is Critical to the Efficient Fluorescence of Cyan Fluorescent Proteins.

PubMed

Gotthard, Guillaume; von Stetten, David; Clavel, Damien; Noirclerc-Savoye, Marjolaine; Royant, Antoine

2017-12-12

ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.

Methods for the investigation of the statistics of the commercial fisheries of the Great Lakes

USGS Publications Warehouse

Hile, Ralph; Duden, William R.

1933-01-01

During the summer and fall of 1939 four experiments were conducted at the Patuxent Research Refuge, Bowie, Maryland, to determine the protein requirements of bobwhite chicks. A total of 816 chicks were used to compare six levels of protein, namely, 22,24,26, 28, 30, and 32 per cent.....From the three standpoints of survival, rate of growth, and efficiency of feed utilization for the first ten weeks of life, the 28 per cent level of protein gave the best results. During the ninth and tenth weeks, the highest efficiency of feed utilization was obtained on the 22 per cent level. The results indicate that after the birds have reached about twothirds of their mature weight, the difference in efficiency between a diet containing 28 per cent of protein and one containing 22 per cent may be small enough to justify, in the interest of economy, the use of a diet containing the lower percentage of protein.

Using radiotelemetry to monitor cardiac response of free-living tule greater white-fronted geese (Anser albifrons elgasi) to human disturbance

USGS Publications Warehouse

Ackerman, Joshua T.; Takekawa, John Y.; Kruse, K.L.; Orthmeyer, D.L.; Yee, J.L.; Ely, Craig R.; Ward, D.H.; Bollinger, K.S.; Mulcahy, D.M.

2004-01-01

During the summer and fall of 1939 four experiments were conducted at the Patuxent Research Refuge, Bowie, Maryland, to determine the protein requirements of bobwhite chicks. A total of 816 chicks were used to compare six levels of protein, namely, 22,24,26, 28, 30, and 32 per cent.....From the three standpoints of survival, rate of growth, and efficiency of feed utilization for the first ten weeks of life, the 28 per cent level of protein gave the best results. During the ninth and tenth weeks, the highest efficiency of feed utilization was obtained on the 22 per cent level. The results indicate that after the birds have reached about twothirds of their mature weight, the difference in efficiency between a diet containing 28 per cent of protein and one containing 22 per cent may be small enough to justify, in the interest of economy, the use of a diet containing the lower percentage of protein.

Non-structural protein 1 of influenza viruses inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, staufen1.

PubMed

Cho, Hana; Ahn, Sang Ho; Kim, Kyoung Mi; Kim, Yoon Ki

2013-07-11

Although non-structural protein 1 (NS1) of influenza viruses is not essential for virulence, this protein is involved in host-virus interactions, viral replication, and translation. In particular, NS1 is known to interact with the host protein, staufen1 (Stau1). This interaction is important for efficient viral replication. However, the underlying molecular mechanism by which NS1 influences the viral life cycle remains obscure. Here, we show using immunoprecipitation and artificial tethering that the N-terminus of NS1, NS1(1-73), interacts with Stau1, blocks the Stau1-Upf1 interaction, and consequently inhibits the efficiency of Stau1-mediated mRNA decay (SMD), but not nonsense-mediatedmRNA decay (NMD). The regulation of SMD efficiency by NS1 may contribute to building a more favorable cellular environment for viral replication. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mean of the typical decoding rates: a new translation efficiency index based on the analysis of ribosome profiling data.

PubMed

Dana, Alexandra; Tuller, Tamir

2014-12-01

Gene translation modeling and prediction is a fundamental problem that has numerous biomedical implementations. In this work we present a novel, user-friendly tool/index for calculating the mean of the typical decoding rates that enables predicting translation elongation efficiency of protein coding genes for different tissue types, developmental stages, and experimental conditions. The suggested translation efficiency index is based on the analysis of the organism's ribosome profiling data. This index could be used for example to predict changes in translation elongation efficiency of lowly expressed genes that usually have relatively low and/or biased ribosomal densities and protein levels measurements, or can be used for example for predicting translation efficiency of new genetically engineered genes. We demonstrate the usability of this index via the analysis of six organisms in different tissues and developmental stages. Distributable cross platform application and guideline are available for download at: http://www.cs.tau.ac.il/~tamirtul/MTDR/MTDR_Install.html. Copyright © 2015 Dana and Tuller.

Efficient siRNA delivery system using carboxilated single-wall carbon nanotubes in cancer treatment.

PubMed

Neagoe, Ioana Berindan; Braicu, Cornelia; Matea, Cristian; Bele, Constantin; Florin, Graur; Gabriel, Katona; Veronica, Chedea; Irimie, Alexandru

2012-08-01

Several functionalized carbon nanotubes have been designed and tested for the purpose of nucleic acid delivery. In this study, the capacity of SWNTC-COOH for siRNA deliverey were investigated delivery in parallel with an efficient commercial system. Hep2G cells were reverse-transfected with 50 nM siRNA (p53 siRNA, TNF-alphasiRNA, VEGFsiRNA) using the siPORT NeoFX (Ambion) transfection agent in paralel with SWNTC-COOH, functionalised with siRNA. The highest level of gene inhibition was observed in the cases treated with p53 siRNA gene; in the case of transfection with siPort, the NeoFX value was 33.8%, while in the case of SWNTC-COOH as delivery system for p53 siRNA was 37.5%. The gene silencing capacity for VEGF was 53.7%, respectively for TNF-alpha 56.7% for siPORT NeoFX delivery systems versus 47.7% (VEGF) and 46.5% (TNF-alpha) for SWNTC-COOH delivery system. SWNTC-COOH we have been showed to have to be an efficient carrier system. The results from the inhibition of gene expresion for both transfection systems were confirmed at protein level. Overall, the lowest mRNA expression was confirmed at protein level, especially in the case of p53 siRNA and TNF-alpha siRNA transfection. Less efficient reduction protein expressions were observed in the case of VEGF siRNA, for both transfection systems at 24 h; only at 48 h, there was a statistically significant reduction of VEGF protein expression. SWCNT-COOH determined an efficient delivery of siRNA. SWNTC-COOH, combined with suitable tumor markers like p53 siRNA, TNFalpha siRNA or VEGF siRNA can be used for the efficient delivery of siRNA.

Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage.

PubMed

Ratanapariyanuch, Kornsulee; Tyler, Robert T; Shim, Youn Young; Reaney, Martin Jt

2012-01-12

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage

PubMed Central

2012-01-01

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes. PMID:22239856

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

PubMed

Nury, Catherine; Redeker, Virginie; Dautrey, Sébastien; Romieu, Anthony; van der Rest, Guillaume; Renard, Pierre-Yves; Melki, Ronald; Chamot-Rooke, Julia

2015-02-03

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications

PubMed Central

Chevrel, Anne; Urvoas, Agathe; de la Sierra-Gallay, Ines Li; Aumont-Nicaise, Magali; Moutel, Sandrine; Desmadril, Michel; Perez, Franck; Gautreau, Alexis; van Tilbeurgh, Herman; Minard, Philippe; Valerio-Lepiniec, Marie

2015-01-01

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes. PMID:26182430

Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants.

PubMed

Nojima, Takahiko; Lin, Angela C; Fujii, Teruo; Endo, Isao

2005-12-01

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.

Gradient gravitational search: An efficient metaheuristic algorithm for global optimization.

PubMed

Dash, Tirtharaj; Sahu, Prabhat K

2015-05-30

The adaptation of novel techniques developed in the field of computational chemistry to solve the concerned problems for large and flexible molecules is taking the center stage with regard to efficient algorithm, computational cost and accuracy. In this article, the gradient-based gravitational search (GGS) algorithm, using analytical gradients for a fast minimization to the next local minimum has been reported. Its efficiency as metaheuristic approach has also been compared with Gradient Tabu Search and others like: Gravitational Search, Cuckoo Search, and Back Tracking Search algorithms for global optimization. Moreover, the GGS approach has also been applied to computational chemistry problems for finding the minimal value potential energy of two-dimensional and three-dimensional off-lattice protein models. The simulation results reveal the relative stability and physical accuracy of protein models with efficient computational cost. © 2015 Wiley Periodicals, Inc.

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli

PubMed Central

Evfratov, Sergey A.; Osterman, Ilya A.; Komarova, Ekaterina S.; Pogorelskaya, Alexandra M.; Rubtsova, Maria P.; Zatsepin, Timofei S.; Semashko, Tatiana A.; Kostryukova, Elena S.; Mironov, Andrey A.; Burnaev, Evgeny; Krymova, Ekaterina; Gelfand, Mikhail S.; Govorun, Vadim M.; Bogdanov, Alexey A.; Dontsova, Olga A.

2017-01-01

Abstract Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs. PMID:27899632

A Proteomic Network for Symbiotic Nitrogen Fixation Efficiency in Bradyrhizobium elkanii.

PubMed

Cooper, Bret; Campbell, Kimberly B; Beard, Hunter S; Garrett, Wesley M; Mowery, Joseph; Bauchan, Gary R; Elia, Patrick

2018-03-01

Rhizobia colonize legumes and reduce N 2 to NH 3 in root nodules. The current model is that symbiotic rhizobia bacteroids avoid assimilating this NH 3 . Instead, host legume cells form glutamine from NH 3 , and the nitrogen is returned to the bacteroid as dicarboxylates, peptides, and amino acids. In soybean cells surrounding bacteroids, glutamine also is converted to ureides. One problem for soybean cultivation is inefficiency in symbiotic N 2 fixation, the biochemical basis of which is unknown. Here, the proteomes of bacteroids of Bradyrhizobium elkanii USDA76 isolated from N 2 fixation-efficient Peking and -inefficient Williams 82 soybean nodules were analyzed by mass spectrometry. Nearly half of the encoded bacterial proteins were quantified. Efficient bacteroids produced greater amounts of enzymes to form Nod factors and had increased amounts of signaling proteins, transporters, and enzymes needed to generate ATP to power nitrogenase and to acquire resources. Parallel investigation of nodule proteins revealed that Peking had no significantly greater accumulation of enzymes needed to assimilate NH 3 than Williams 82. Instead, efficient bacteroids had increased amounts of enzymes to produce amino acids, including glutamine, and to form ureide precursors. These results support a model for efficient symbiotic N 2 fixation in soybean where the bacteroid assimilates NH 3 for itself.

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Hsu, Chuan-Chih; Xue, Liang; Arrington, Justine V.; Wang, Pengcheng; Paez Paez, Juan Sebastian; Zhou, Yuan; Zhu, Jian-Kang; Tao, W. Andy

2017-06-01

Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. [Figure not available: see fulltext.

Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics

PubMed Central

2011-01-01

Background High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. Results The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. Conclusions The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers. PMID:21554704

Growth, oxygen consumption, and protein and RNA synthesis rates in the yolk sac larvae of the African catfish (Clarias gariepinus).

PubMed

Smith, Richard W; Ottema, Colin

2006-03-01

Rapidly growing African catfish yolk sac larvae were investigated during the first 22 h after hatching. Body compartment protein concentration increased fourfold yet oxygen consumption remained constant (mean=21.3 +/- 3.2 nmol O2 mg(-1) protein h(-1)), suggesting fast growth results mainly from yolk sac protein absorption. The protein synthesis rates at 1-2 and 5-6 h also equaled the highest conceivable rates of muscle protein synthesis; 11.6-11.9% and 7.4-7.9% day(-1), respectively. Therefore the corresponding energetic costs of protein synthesis were almost the theoretical minimum; 13.0 +/- 1.7-16.3 +/- 2.8 micromol O2 mg(-1) protein synthesised. Total protein synthesis expenditure (74.5-77.7 micromol O2 g(-1) protein h(-1)) was also less than other yolk sac larvae. These protein synthesis rates resulted from high RNA concentrations (113.2 +/- 3.4 microg RNA mg(-1) protein) and were also correlated with RNA translational efficiency. High translational efficiency (1 h; 1.2+/-0.1 mg protein synthesised microg(-1) RNA day(-1)) equaled high synthesis rate (36.8 +/- 5.4 microg RNA microg(-1) DNA day(-1)) and both declined over 22 h. This investigation suggests rapid growth combines growth efficiency and compensatory energy partitioning. This accommodates the ontogenetic and phylogenetic standpoints imposed by energy budget limitations.

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

PubMed

Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

2000-02-01

Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE PAGES

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo; ...

2016-05-17

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Mixotrophic cultivation of Chlorella for local protein production using agro-food by-products.

PubMed

Salati, Silvia; D'Imporzano, Giuliana; Menin, Barbara; Veronesi, Davide; Scaglia, Barbara; Abbruscato, Pamela; Mariani, Paola; Adani, Fabrizio

2017-04-01

A local strain of Chlorella vulgaris was cultivated by using cheese whey (CW), white wine lees (WL) and glycerol (Gly), coming from local agro-industrial activities, as C sources (2.2gCL -1 ) to support algae production under mixotrophic conditions in Lombardy. In continuous mode, Chlorella increased biomass production compared with autotrophic conditions by 1.5-2 times, with the best results obtained for the CW substrate, i.e. 0.52gL -1 d -1 of algal biomass vs. 0.24gL -1 d -1 of algal biomass for autotrophic conditions, and protein content for both conditions adopted close to 500gkg -1 DM. Mixotrophic conditions gave a much higher energy recovery efficiency (EF) than autotrophic conditions, i.e. organic carbon energy efficiency (EF oc ) of 32% and total energy efficiency (Ef t ) of 8%, respectively, suggesting the potential for the culture of algae as a sustainable practice to recover efficiently waste-C and a means of local protein production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location.

PubMed

Goedhart, Joachim; van Unen, Jakobus; Adjobo-Hermans, Merel J W; Gadella, Theodorus W J

2013-01-01

The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

PubMed Central

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

PubMed Central

Dewari, Pooran Singh; Southgate, Benjamin; Mccarten, Katrina; Monogarov, German; O'Duibhir, Eoghan; Quinn, Niall; Tyrer, Ashley; Leitner, Marie-Christin; Plumb, Colin; Kalantzaki, Maria; Blin, Carla; Finch, Rebecca; Bressan, Raul Bardini; Morrison, Gillian; Jacobi, Ashley M; Behlke, Mark A; von Kriegsheim, Alex; Tomlinson, Simon; Krijgsveld, Jeroen

2018-01-01

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells. PMID:29638216

Translation efficiency is determined by both codon bias and folding energy

PubMed Central

Tuller, Tamir; Waldman, Yedael Y.; Kupiec, Martin; Ruppin, Eytan

2010-01-01

Synonymous mutations do not alter the protein produced yet can have a significant effect on protein levels. The mechanisms by which this effect is achieved are controversial; although some previous studies have suggested that codon bias is the most important determinant of translation efficiency, a recent study suggested that mRNA folding at the beginning of genes is the dominant factor via its effect on translation initiation. Using the Escherichia coli and Saccharomyces cerevisiae transcriptomes, we conducted a genome-scale study aiming at dissecting the determinants of translation efficiency. There is a significant association between codon bias and translation efficiency across all endogenous genes in E. coli and S. cerevisiae but no association between folding energy and translation efficiency, demonstrating the role of codon bias as an important determinant of translation efficiency. However, folding energy does modulate the strength of association between codon bias and translation efficiency, which is maximized at very weak mRNA folding (i.e., high folding energy) levels. We find a strong correlation between the genomic profiles of ribosomal density and genomic profiles of folding energy across mRNA, suggesting that lower folding energies slow down the ribosomes and decrease translation efficiency. Accordingly, we find that selection forces act near uniformly to decrease the folding energy at the beginning of genes. In summary, these findings testify that in endogenous genes, folding energy affects translation efficiency in a global manner that is not related to the expression levels of individual genes, and thus cannot be detected by correlation with their expression levels. PMID:20133581

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

PubMed

Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

2016-11-15

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

Encapsulation performance of layer-by-layer microcapsules for proteins.

PubMed

De Temmerman, Marie-Luce; Demeester, Jo; De Vos, Filip; De Smedt, Stefaan C

2011-04-11

This study reports on the encapsulation efficiency of proteins in dextran sulfate/poly-L-arginine-based microcapsules, fabricated via layer-by-layer assembly (LbL). For this purpose, radiolabeled proteins are entrapped in CaCO(3) microparticles, followed by LbL coating of the CaCO(3) cores and subsequent dissolving of the CaCO(3) using EDTA. To allow to improve protein encapsulation in LbL microcapsules, we studied all steps in the preparation of the microcapsules where loss of protein load might occur. The encapsulation efficiency of proteins in LbL microcapsules turns out to be strongly dependent on both the charge and molecular weight of the protein as well as on the number of polyelectrolyte bilayers the microcapsules consist of.

Comparative shoot proteome analysis of two potato (Solanum tuberosum L.) genotypes contrasting in nitrogen deficiency responses in vitro.

PubMed

Meise, Philipp; Jozefowicz, Anna Maria; Uptmoor, Ralf; Mock, Hans-Peter; Ordon, Frank; Schum, Annegret

2017-08-23

Aiming at a better understanding of the physiological and biochemical background of nitrogen use efficiency, alterations in the shoot proteome under N-deficiency were investigated in two contrasting potato genotypes grown in vitro with 60 and 7.5mM N, respectively. A gel based proteomic approach was applied to identify candidate proteins associated with genotype specific responses to N-deficiency. 21% of the detected proteins differed in abundance between the two genotypes. Between control and N-deficiency conditions 19.5% were differentially accumulated in the sensitive and 15% in the tolerant genotype. 93% of the highly N-deficiency responsive proteins were identified by MALDI TOF/TOF mass spectrometry. The major part was associated with photosynthesis, carbohydrate metabolism, stress response and regulation. Differential accumulation of enzymes involved in the Calvin cycle and glycolysis suggest activation of alternative carbohydrate pathways. In the tolerant genotype, increased abundance under N-deficiency was also found for enzymes involved in chlorophyll synthesis and stability of enzymes, which increase photosynthetic carbon fixation efficiency. Out of a total of 106 differentially abundant proteins, only eight were detected in both genotypes. Our findings suggest that mutually responsive proteins reflect universal stress responses while adaptation to N-deficiency in metabolic pathways is more genotype specific. Nitrogen losses from arable farm land considerably contribute to environmental pollution. In potato, this is a special problem due cultivation on light soils, irrigation and the shallow root system. Therefore, breeding of cultivars with improved nitrogen use efficiency and stable yields under reduced N fertilization is an important issue. Knowledge of genotype dependent adaptation to N-deficiency at the proteome level can help to understand regulation of N efficiency and development of N-efficient cultivars. Copyright © 2017 Elsevier B.V. All rights reserved.

Liposome-chaperoned cell-free synthesis for the design of proteoliposomes: Implications for therapeutic delivery.

PubMed

Lu, Mei; Zhao, Xiaoyun; Xing, Haonan; Xun, Zhe; Yang, Tianzhi; Cai, Cuifang; Wang, Dongkai; Ding, Pingtian

2018-04-03

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery. Copyright © 2018. Published by Elsevier Ltd.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Min Sun; Biosystems and Bioengineering Program, University of Science and Technology; Mun, Ji-Young

2013-07-19

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineeredmore » the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.« less

Packaging signals in single-stranded RNA viruses: nature's alternative to a purely electrostatic assembly mechanism.

PubMed

Stockley, Peter G; Twarock, Reidun; Bakker, Saskia E; Barker, Amy M; Borodavka, Alexander; Dykeman, Eric; Ford, Robert J; Pearson, Arwen R; Phillips, Simon E V; Ranson, Neil A; Tuma, Roman

2013-03-01

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA-coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

PubMed Central

Tkachenko, Anastasiya; Richter, Vladimir

2017-01-01

Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes.

PubMed

Liu, Yangyang; Han, Xiao; Yuan, Junting; Geng, Tuoyu; Chen, Shihao; Hu, Xuming; Cui, Isabelle H; Cui, Hengmi

2017-04-07

The type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of PPP1R12C , NSUN2 (protein-coding genes), and MALAT1 (non-protein-coding gene). Both NeoR and PuroR were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

Improvement of proteolytic efficiency towards low-level proteins by an antifouling surface of alumina gel in a microchannel.

PubMed

Liu, Yun; Wang, Huixiang; Liu, Qingping; Qu, Haiyun; Liu, Baohong; Yang, Pengyuan

2010-11-07

A microfluidic reactor has been developed for rapid enhancement of protein digestion by constructing an alumina network within a poly(ethylene terephthalate) (PET) microchannel. Trypsin is stably immobilized in a sol-gel network on the PET channel surface after pretreatment, which produces a protein-resistant interface to reduce memory effects, as characterized by X-ray fluorescence spectrometry and electroosmotic flow. The gel-derived network within a microchannel provides a large surface-to-volume ratio stationary phase for highly efficient proteolysis of proteins existing both at a low level and in complex extracts. The maximum reaction rate of the encapsulated trypsin reactor, measured by kinetic analysis, is much faster than in bulk solution. Due to the microscopic confinement effect, high levels of enzyme entrapment and the biocompatible microenvironment provided by the alumina gel network, the low-level proteins can be efficiently digested using such a microreactor within a very short residence time of a few seconds. The on-chip microreactor is further applied to the identification of a mixture of proteins extracted from normal mouse liver cytoplasm sample via integration with 2D-LC-ESI-MS/MS to show its potential application for large-scale protein identification.

Controlling chitosan-based encapsulation for protein and vaccine delivery

PubMed Central

Koppolu, Bhanu prasanth; Smith, Sean G.; Ravindranathan, Sruthi; Jayanthi, Srinivas; Kumar, Thallapuranam K.S.; Zaharoff, David A.

2014-01-01

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation-coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, was inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters. PMID:24560459

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

PubMed

Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

2010-05-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

PubMed Central

2015-01-01

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community. PMID:25121619

Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

PubMed

Hui, James Z; Tsourkas, Andrew

2014-09-17

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine.

PubMed

Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

2015-01-01

Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes.

PubMed

Li, Xiang; Yang, Weijing; Zou, Yan; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan

2015-12-28

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157-175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1-24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine

PubMed Central

Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

2015-01-01

Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable. PMID:26208298

Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

PubMed

Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

2014-01-01

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

PubMed

Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

2016-05-01

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

PubMed

Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

2017-03-01

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidating the mechanism of protein water channels by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Grubmuller, Helmut

2004-03-01

Aquaporins are highly selective water channels. Molecular dynamics simulations of multiple water permeation events correctly predict the measured rate and explain at the atomic level why these membrane channels are so efficient, while blocking other small molecules, ions, and even protons. High efficiency is achieved through a carefully tailored balance of hydrogen bonds that the protein substitutes for the bulk interactions; selectivity is achieved mainly by electrostatic barriers.

Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris.

PubMed

Theron, Chrispian W; Berrios, Julio; Delvigne, Frank; Fickers, Patrick

2018-01-01

The methylotrophic yeast Komagataella (Pichia) pastoris has become one of the most utilized cell factories for the production of recombinant proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris-based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of production. In this review, new perspectives are detailed regarding a multi-faceted approach to recombinant protein production processes by P. pastoris; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.

Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yuqi; Wang, Jinan; Shao, Qiang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn

2015-03-28

The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much lessmore » computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.« less

Action-FRET of a Gaseous Protein

NASA Astrophysics Data System (ADS)

Daly, Steven; Knight, Geoffrey; Halim, Mohamed Abdul; Kulesza, Alexander; Choi, Chang Min; Chirot, Fabien; MacAleese, Luke; Antoine, Rodolphe; Dugourd, Philippe

2017-01-01

Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question—and thus further develop mass spectrometry as a tool for structural biology—structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state—where a systematic decrease is observed—and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization.

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

PubMed

Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Comparison of sludge digestion under aerobic and anaerobic conditions with a focus on the degradation of proteins at mesophilic temperature.

PubMed

Shao, Liming; Wang, Tianfeng; Li, Tianshui; Lü, Fan; He, Pinjing

2013-07-01

Aerobic and anaerobic digestion are popular methods for the treatment of waste activated sludge. However, the differences in degradation of sludge during aerobic and anaerobic digestion remain unclear. In this study, the sludge degradation during aerobic and anaerobic digestion was investigated at mesophilic temperature, focused on protein based on the degradation efficiency and degree of humification. The duration of aerobic and anaerobic digestion was about 90 days. The final degradation efficiency of volatile solid was 66.1 ± 1.6% and 66.4 ± 2.4% under aerobic and anaerobic conditions, respectively. The final degradation efficiency of protein was 67.5 ± 1.4% and 65.1 ± 2.6% under aerobic and anaerobic conditions, respectively. The degradation models of volatile solids were consistent with those of protein under both aerobic and anaerobic conditions. The solubility of protein under aerobic digestion was greater than that under anaerobic digestion. Moreover, the humification index of dissolved organic matter of aerobic digestion was greater than that during anaerobic digestion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Land-based production of animal protein: impacts, efficiency, and sustainability.

PubMed

Wu, Guoyao; Bazer, Fuller W; Cross, H Russell

2014-11-01

Land-based production of high-quality protein by livestock and poultry plays an important role in improving human nutrition, growth, and health, as well as economical and social developments worldwide. With exponential growth of the global population and marked rises in meat consumption per capita, demands for animal-source protein are expected to increase by 72% between 2013 and 2050. This raises concerns about the sustainability and environmental impacts of animal agriculture. An attractive solution to meeting the increasing needs for animal products and mitigating undesired effects of agricultural practices is to enhance the efficiency of animal growth, reproduction, and lactation. Breeding techniques may help achieve this goal, but have only met with limited success. A promising, mechanism-based approach is to optimize the proportion and amounts of amino acids in diets for maximizing whole-body protein synthesis and feed efficiency. Improvements in farm animal productivity will not only decrease the contamination of soils, groundwater, and air by excessive manure, but will also help sustain animal agriculture to produce high-quality protein for the expanding population in the face of diminishing resources. © 2014 New York Academy of Sciences.

Compound Synthesis or Growth and Development of Roots/Stomata Regulate Plant Drought Tolerance or Water Use Efficiency/Water Uptake Efficiency.

PubMed

Meng, Lai-Sheng

2018-04-11

Water is crucial to plant growth and development because it serves as a medium for all cellular functions. Thus, the improvement of plant drought tolerance or water use efficiency/water uptake efficiency is important in modern agriculture. In this review, we mainly focus on new genetic factors for ameliorating drought tolerance or water use efficiency/water uptake efficiency of plants and explore the involvement of these genetic factors in the regulation of improving plant drought tolerance or water use efficiency/water uptake efficiency, which is a result of altered stomata density and improving root systems (primary root length, hair root growth, and lateral root number) and enhanced production of osmotic protectants, which is caused by transcription factors, proteinases, and phosphatases and protein kinases. These results will help guide the synthesis of a model for predicting how the signals of genetic and environmental stress are integrated at a few genetic determinants to control the establishment of either water use efficiency or water uptake efficiency. Collectively, these insights into the molecular mechanism underpinning the control of plant drought tolerance or water use efficiency/water uptake efficiency may aid future breeding or design strategies to increase crop yield.

Growth efficiency in transgenic tilapia (Oreochromis sp.) carrying a single copy of an homologous cDNA growth hormone.

PubMed

Martínez, R; Juncal, J; Zaldívar, C; Arenal, A; Guillén, I; Morera, V; Carrillo, O; Estrada, M; Morales, A; Estrada, M P

2000-01-07

Growth hormone (GH) has been shown to have a profound impact on fish physiology and metabolism. However, detailed studies in transgenic fish have not been conducted. We have characterized the food conversion efficiency, protein profile, and biochemical correlates of growth rate in transgenic tilapia expressing the tilapia GH cDNA under the control of human cytomegalovirus regulatory sequences. Transgenic tilapia exhibited about 3.6-fold less food consumption than nontransgenic controls (P < 0.001). The food conversion efficiency was significantly (P < 0.05) higher (290%) in transgenic tilapia (2.3 +/- 0.4) than in the control group (0.8 +/- 0.2). Efficiency of growth, synthesis retention, anabolic stimulation, and average protein synthesis were higher in transgenic than in nontransgenic tilapia. Distinctive metabolic differences were found in transgenic juvenile tilapia. We had found differences in hepatic glucose, and in agreement with previous results we observed differences in the level of enzymatic activities in target organs. We conclude that GH-transgenic juvenile tilapia show altered physiological and metabolic conditions and are biologically more efficient. Copyright 2000 Academic Press.

Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

PubMed

Xia, Yuan-Jun; Xia, Hong; Chen, Ling; Ying, Qing-Shui; Yu, Xiang; Li, Li-Hua; Wang, Jian-Hua; Zhang, Ying

2018-04-01

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

PubMed

Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

2016-02-01

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

PubMed Central

Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

2017-01-01

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

Liver plasma membranes: an effective method to analyze membrane proteome.

PubMed

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.

Review: Optimizing ruminant conversion of feed protein to human food protein.

PubMed

Broderick, G A

2017-11-20

Ruminant livestock have the ability to produce high-quality human food from feedstuffs of little or no value for humans. Balanced essential amino acid composition of meat and milk from ruminants makes those protein sources valuable adjuncts to human diets. It is anticipated that there will be increasing demand for ruminant proteins in the future. Increasing productivity per animal dilutes out the nutritional and environmental costs of maintenance and rearing dairy animals up to production. A number of nutritional strategies improve production per animal such as ration balancing in smallholder operations and small grain supplements to ruminants fed high-forage diets. Greenhouse gas emission intensity is reduced by increased productivity per animal; recent research has developed at least one effective inhibitor of methane production in the rumen. There is widespread over-feeding of protein to dairy cattle; milk and component yields can be maintained, and sometimes even increased, at lower protein intake. Group feeding dairy cows according to production and feeding diets higher in rumen-undegraded protein can improve milk and protein yield. Supplementing rumen-protected essential amino acids will also improve N efficiency in some cases. Better N utilization reduces urinary N, which is the most environmentally unstable form of excretory N. Employing nutritional models to more accurately meet animal requirements improves nutrient efficiency. Although smallholder enterprises, which are concentrated in tropical and semi-tropical regions of developing countries, are subject to different economic pressures, nutritional biology is similar at all production levels. Rather than milk volume, nutritional strategies should maximize milk component yield, which is proportional to market value as well as food value when milk nutrients are consumed directly by farmers and their families. Moving away from Holsteins toward smaller breeds such as Jerseys, Holstein-Jersey crosses or locally adapted breeds (e.g. Vechur) would also reduce lactose production and improve metabolic, environmental and economic efficiencies. Forages containing condensed tannins or polyphenol oxidase enzymes have reduced rumen protein degradation; ruminants capture this protein more efficiently for meat and milk. Although these forages generally have lower yields and persistence, genetic modification would allow insertion of these traits into more widely cultivated forages. Ruminants will retain their niches because of their ability to produce valuable human food from low value feedstuffs. Employing these emerging strategies will allow improved productive efficiency of ruminants in both developing and developed countries.

Room-temperature synthesis of core-shell structured magnetic covalent organic frameworks for efficient enrichment of peptides and simultaneous exclusion of proteins.

PubMed

Lin, Guo; Gao, Chaohong; Zheng, Qiong; Lei, Zhixian; Geng, Huijuan; Lin, Zian; Yang, Huanghao; Cai, Zongwei

2017-03-28

Core-shell structured magnetic covalent organic frameworks (Fe 3 O 4 @COFs) were synthesized via a facile approach at room temperature. Combining the advantages of high porosity, magnetic responsiveness, chemical stability and selectivity, Fe 3 O 4 @COFs can serve as an ideal absorbent for the highly efficient enrichment of peptides and the simultaneous exclusion of proteins from complex biological samples.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shaw, Debra J.; Morse, Robert; Todd, Adrian G.

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import,more » they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.« less

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Efficient Conformational Sampling in Explicit Solvent Using a Hybrid Replica Exchange Molecular Dynamics Method

DTIC Science & Technology

2011-12-01

REMD while reproducing the energy landscape of explicit solvent simulations . ’ INTRODUCTION Molecular dynamics (MD) simulations of proteins can pro...Mongan, J.; McCammon, J. A. Accelerated molecular dynamics : a promising and efficient simulation method for biomolecules. J. Chem. Phys. 2004, 120 (24...Chemical Theory and Computation ARTICLE (8) Abraham,M. J.; Gready, J. E. Ensuringmixing efficiency of replica- exchange molecular dynamics simulations . J

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells

PubMed Central

Chow‐Shi‐Yée, Mélanie; Briard, Jennie G.; Grondin, Mélanie; Averill‐Bates, Diana A.

2016-01-01

Abstract Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post‐thaw. We are developing new technology where plant proteins are used to substitute the commonly‐used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI‐2 (ice recrystallization inhibition), TaBAS1 (2‐Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin‐secreting INS832/13 cells. This study shows that TaIRI‐2 and TaENO are internalized during the freeze–thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The “splat cooling” method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI‐2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI‐2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin‐secreting cells, and possibly other cell types. TaENO does not have typical ice‐binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. PMID:26889747

Highly efficient delivery of siRNA to a heart transplant model by a novel cell penetrating peptide-dsRNA binding domain.

PubMed

Li, Hua; Zheng, Xiangtao; Koren, Viktoria; Vashist, Yogesh Kumar; Tsui, Tung Yu

2014-07-20

Small interfering RNAs (siRNAs) delivery remains a bottleneck for RNA interference (RNAi) - based therapies in the clinic. In the present study, a fusion protein with two cell-penetrating peptides (CPP), Hph1-Hph1, and a double-stranded RNA binding domain (dsRBD), was constructed for the siRNA delivery: dsRBD was designed to bind siRNA, and CPP would subsequently transport the dsRBD/siRNA complex into cells. We assessed the efficiency of the fusion protein, Hph1-Hph1-dsRBD, as a siRNA carrier. Calcium-condensed effects were assessed on GAPDH and green fluorescent protein (GFP) genes by western blot, real time polymerase chain reaction (RT-PCR), and flow cytometry analysis in vitro. Evaluations were also made in an in vivo heart transplantation model. The results demonstrated that the fusion protein, Hph1-Hph1-dsRBD, is highly efficient at delivering siRNA in vitro, and exhibits efficiency on GAPDH and GFP genes similar to or greater than lipofectamine. Interestingly, the calcium-condensed effects dramatically enhanced cellular uptake of the protein-siRNA complex. In vivo, Hph1-Hph1-dsRBD transferred and distributed ^ targeted siRNA throughout the whole mouse heart graft. Together, these results indicate that Hph1-Hph1-dsRBD has potential as an siRNA carrier for applications in the clinic or in biomedical research. Copyright © 2014 Elsevier B.V. All rights reserved.

Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Hong-Zhang; Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC; Wu, Carol P.

2011-02-11

Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their abilitymore » to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.« less

Maximizing efficiency of rumen microbial protein production

PubMed Central

Hackmann, Timothy J.; Firkins, Jeffrey L.

2015-01-01

Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen. PMID:26029197

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

PubMed Central

Fan, Chenguang; Xiong, Hai; Reynolds, Noah M.; Söll, Dieter

2015-01-01

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNAPyl to create tRNAPyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (Nϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNAPyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNAPyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNAPyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNAPyl-opt should be an excellent replacement of wild-type tRNAPyl for future ncAA incorporation by PylRS enzymes. PMID:26250114

High-Efficiency Synthesis of Human α-Endorphin and Magainin in the Erythrocytes of Transgenic Mice: A Production System for Therapeutic Peptides

NASA Astrophysics Data System (ADS)

Sharma, Ajay; Khoury-Christianson, Anastasia M.; White, Steven P.; Dhanjal, Nirpal K.; Huang, Wen; Paulhiac, Clara; Friedman, Eric J.; Manjula, Belur N.; Kumar, Ramesh

1994-09-01

Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptide as an extension at the C end of human α-globin. A protein cleavage site is inserted between the C end of the α-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer α-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain healthy, even when the fusion protein is expressed at >25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.

A robust heteronuclear dipolar recoupling method comparable to TEDOR for proteins in magic-angle spinning solid-state NMR

NASA Astrophysics Data System (ADS)

Zhang, Zhengfeng; Li, Jianping; Chen, Yanke; Xie, Huayong; Yang, Jun

2017-12-01

In this letter, we propose a robust heteronuclear dipolar recoupling method for proteins in magic-angle spinning (MAS) solid-state NMR. This method is as simple, robust and efficient as the well-known TEDOR in the aspect of magnetization transfer between 15N and 13C. Deriving from our recent band-selective dual back-to-back pulses (DBP) (Zhang et al., 2016), this method uses new phase-cycling schemes to realize broadband DBP (Bro-DBP). For broadband 15N-13C magnetization transfer (simultaneous 15N → 13C‧ and 15N → 13Cα), Bro-DBP has almost the same 15N → 13Cα efficiency while offers 30-40% enhancement on 15N → 13C‧ transfer, compared to TEDOR. Besides, Bro-DBP can also be used as a carbonyl (13C‧)-selected method, whose 15N → 13C‧ efficiency is up to 1.7 times that of TEDOR and is also higher than that of band-selective DBP. The performance of Bro-DBP is demonstrated on the N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (fMLF) peptide and the U-13C, 15N labeled β1 immunoglobulin binding domain of protein G (GB1) microcrystalline protein. Since Bro-DBP is as robust, simple and efficient as TEDOR, we believe it is very useful for protein studies in MAS solid-state NMR.

Balanced Codon Usage Optimizes Eukaryotic Translational Efficiency

PubMed Central

Qian, Wenfeng; Yang, Jian-Rong; Pearson, Nathaniel M.; Maclean, Calum; Zhang, Jianzhi

2012-01-01

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology. PMID:22479199

Social networks in primates: smart and tolerant species have more efficient networks.

PubMed

Pasquaretta, Cristian; Levé, Marine; Claidière, Nicolas; van de Waal, Erica; Whiten, Andrew; MacIntosh, Andrew J J; Pelé, Marie; Bergstrom, Mackenzie L; Borgeaud, Christèle; Brosnan, Sarah F; Crofoot, Margaret C; Fedigan, Linda M; Fichtel, Claudia; Hopper, Lydia M; Mareno, Mary Catherine; Petit, Odile; Schnoell, Anna Viktoria; di Sorrentino, Eugenia Polizzi; Thierry, Bernard; Tiddi, Barbara; Sueur, Cédric

2014-12-23

Network optimality has been described in genes, proteins and human communicative networks. In the latter, optimality leads to the efficient transmission of information with a minimum number of connections. Whilst studies show that differences in centrality exist in animal networks with central individuals having higher fitness, network efficiency has never been studied in animal groups. Here we studied 78 groups of primates (24 species). We found that group size and neocortex ratio were correlated with network efficiency. Centralisation (whether several individuals are central in the group) and modularity (how a group is clustered) had opposing effects on network efficiency, showing that tolerant species have more efficient networks. Such network properties affecting individual fitness could be shaped by natural selection. Our results are in accordance with the social brain and cultural intelligence hypotheses, which suggest that the importance of network efficiency and information flow through social learning relates to cognitive abilities.

Social networks in primates: smart and tolerant species have more efficient networks

PubMed Central

Pasquaretta, Cristian; Levé, Marine; Claidière, Nicolas; van de Waal, Erica; Whiten, Andrew; MacIntosh, Andrew J. J.; Pelé, Marie; Bergstrom, Mackenzie L.; Borgeaud, Christèle; Brosnan, Sarah F.; Crofoot, Margaret C.; Fedigan, Linda M.; Fichtel, Claudia; Hopper, Lydia M.; Mareno, Mary Catherine; Petit, Odile; Schnoell, Anna Viktoria; di Sorrentino, Eugenia Polizzi; Thierry, Bernard; Tiddi, Barbara; Sueur, Cédric

2014-01-01

Network optimality has been described in genes, proteins and human communicative networks. In the latter, optimality leads to the efficient transmission of information with a minimum number of connections. Whilst studies show that differences in centrality exist in animal networks with central individuals having higher fitness, network efficiency has never been studied in animal groups. Here we studied 78 groups of primates (24 species). We found that group size and neocortex ratio were correlated with network efficiency. Centralisation (whether several individuals are central in the group) and modularity (how a group is clustered) had opposing effects on network efficiency, showing that tolerant species have more efficient networks. Such network properties affecting individual fitness could be shaped by natural selection. Our results are in accordance with the social brain and cultural intelligence hypotheses, which suggest that the importance of network efficiency and information flow through social learning relates to cognitive abilities. PMID:25534964

Rumen microbial protein synthesis and nitrogen efficiency as affected by tanniferous and non-tanniferous forage legumes incubated individually or together in Rumen Simulation Technique.

PubMed

Grosse Brinkhaus, Anja; Bee, Giuseppe; Schwarm, Angela; Kreuzer, Michael; Dohme-Meier, Frigga; Zeitz, Johanna O

2018-03-01

A limited availability of microbial protein can impair productivity in ruminants. Ruminal nitrogen efficiency might be optimised by combining high-quality forage legumes such as red clover (RC), which has unfavourably high ruminal protein degradability, with tanniferous legumes like sainfoin (SF) and birdsfoot trefoil (BT). Silages from SF and from BT cultivars [Bull (BB) and Polom (BP)] were incubated singly or in combination with RC using the Rumen Simulation Technique (n = 6). The tanniferous legumes, when compared to RC, changed the total short-chain fatty acid profile by increasing propionate proportions at the expense of butyrate. Silage from SF contained the most condensed tannins (CTs) (136 g CT kg -1 dry matter) and clearly differed in various traits from the BT and RC silages. The apparent nutrient degradability (small with SF), microbial protein synthesis, and calculated content of potentially utilisable crude protein (large with SF) indicated that SF had the greatest efficiency in ruminal protein synthesis. The effects of combining SF with RC were mostly linear. The potential of sainfoin to improve protein supply, demonstrated either individually or in combination with a high-performance forage legume, indicates its potential usefulness in complementing protein-deficient ruminant diets and high-quality forages rich in rumen-degradable protein. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

PubMed

Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

2017-08-22

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

PubMed Central

Tani, Hideki; Limn, Chang Kwang; Yap, Chan Choo; Onishi, Masayoshi; Nozaki, Masami; Nishimune, Yoshitake; Okahashi, Nobuo; Kitagawa, Yoshinori; Watanabe, Rie; Mochizuki, Rika; Moriishi, Kohji; Matsuura, Yoshiharu

2003-01-01

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy. PMID:12941888

Construction of a highly efficient display system for baculovirus and its application on multigene co-display.

PubMed

Zheng, Hao; Wang, Xiong; Ren, Feifei; Zou, Shenglong; Feng, Min; Xu, Liangliang; Yao, Lunguang; Sun, Jingchen

2018-06-19

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. PMID:26792745

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

An improved design of spiral tube assembly for separation of proteins by high-speed counter-current chromatography.

PubMed

Dasarathy, Dhweeja; Ito, Yoichiro

2015-10-30

A new spiral tube assembly was designed to improve the column capacity and partition efficiency for protein separation. This spiral tube assembly has greater column capacity than the original tubing because of an increase in radial grooves from 4 to 12 to accommodate more spiral layers and 12 narrow spots instead of 4 in each circular loop to interrupt the laminar flow that causes sample band broadening. Standard PTFE tubing (1.6mm ID) and the modified flat-twisted tubing were used as the separation column. The performances of both assemblies were compared for separating three stable test proteins including cytochrome c, myoglobin, and lysozyme using a two phase aqueous-aqueous solvent system composed of polyethylene glycol 1000 (12.5% w/w) and dibasic potassium phosphate (12.5% w/w). All samples were run at 1, 2, 3, and 5mL/min at both 800rpm and 1000rpm. The separation of these three protein samples produced high stationary phase retentions at 1, 2, and 3mL/min, yet separated efficiently at 5mL/min in 40min. After comparing the separation efficiency in terms of the peak resolutions, theoretical plate numbers, and separation times, it was determined that the flat-twisted tubing was more effective in separating these protein samples. In order to validate the efficacy of this novel assembly, a mixture of five protein samples (cytochrome c, myoglobin, ovalbumin, lysozyme, and hemoglobin) were separated, under the optimal conditions established with these three protein samples, at 1mL/min with a revolution speed of 1000rpm. There were high stationary phase retentions of around 60%, with effective separations, demonstrating the efficiency of the flat-twisted spiral tube assembly. The separation time of 6h was a limitation but can potentially be shortened by improving the strength of the column that will permit an increase in revolution speed and flow rate. This novel spiral separation column will allow rapid and efficient separation of mixtures with high yield of the constituent components. Published by Elsevier B.V.

The dependence of efficiency of transmembrane molecular transfer using electroporation on medium viscosity.

PubMed

Sungailaitė, Sandra; Ruzgys, Paulius; Šatkauskienė, Ingrida; Čepurnienė, Karolina; Šatkauskas, Saulius

2015-01-01

In the present study, we aimed to evaluate the efficiency of drug and gene electrotransfer into cells in vitro depending on medium viscosity. Experiments were performed using Chinese hamster ovary cells. Efficiency of molecular electrotransfer depending of medium viscosity was evaluated using two different electroporation conditions: a high-voltage (HV) pulse and a combination of a high-voltage pulse and a low-voltage pulse (HV + LV). To evaluate the efficiency of molecular electrotransfer, anticancer drug bleomycin and two different plasmids coding for green fluorescent protein and luciferase were used. We found that a slight increase in medium viscosity from 1.3-1.4 mPa·s significantly decreased the transfection efficiency, both in terms of transfected cells and total protein production, which was abolished completely with an increase in medium viscosity to 6.1 mPa·s. Notably, at this medium viscosity, electrotransfer of the small anticancer drug was still efficient. Using HV and HV + LV pulse combinations, we showed that a decrease of DNA electrotransfer, especially at lower medium viscosities, can be compensated for by the LV pulse to some extent. On the other hand, the addition of the LV pulse after the HV pulse did not have any positive effect on the efficiency of bleomycin electrotransfer. These findings demonstrate that transfection is very susceptible to medium viscosity and highlights the importance of the electrophoretic component in experiments when a considerable transfection level is needed. Copyright © 2015 John Wiley & Sons, Ltd.

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

PubMed Central

Brindley, Melinda A.; Plattet, Philippe; Plemper, Richard Karl

2014-01-01

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. PMID:25157143

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain.

PubMed

Brindley, Melinda A; Plattet, Philippe; Plemper, Richard Karl

2014-09-09

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry.

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

PubMed

Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

2017-06-27

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

Efficient light-harvesting using non-carbonyl carotenoids: Energy transfer dynamics in the VCP complex from Nannochloropsis oceanica.

PubMed

Keşan, Gürkan; Litvín, Radek; Bína, David; Durchan, Milan; Šlouf, Václav; Polívka, Tomáš

2016-04-01

Violaxanthin-chlorophyll a protein (VCP) from Nannochloropsis oceanica is a Chl a-only member of the LHC family of light-harvesting proteins. VCP binds carotenoids violaxanthin (Vio), vaucheriaxanthin (Vau), and vaucheriaxanthin-ester (Vau-ester). Here we report on energy transfer pathways in the VCP complex. The overall carotenoid-to-Chla energy transfer has efficiency over 90%. Based on their energy transfer properties, the carotenoids in VCP can be divided into two groups; blue carotenoids with the lowest energy absorption band around 480nm and red carotenoids with absorption extended up to 530nm. Both carotenoid groups transfer energy efficiently from their S2 states, reaching efficiencies of ~70% (blue) and ~60% (red). The S1 pathway, however, is efficient only for the red carotenoid pool for which two S1 routes characterized by 0.33 and 2.4ps time constants were identified. For the blue carotenoids the S1-mediated pathway is represented only by a minor route likely involving a hot S1 state. The relaxed S1 state of blue carotenoids decays to the ground state within 21ps. Presence of a fraction of non-transferring red carotenoids with the S1 lifetime of 13ps indicates some specific carotenoid-protein interaction that must shorten the intrinsic S1 lifetime of Vio and/or Vau whose S1 lifetimes in methanol are 26 and 29ps, respectively. The VCP complex from N. oceanica is the first example of a light-harvesting complex binding only non-carbonyl carotenoids with carotenoid-to-chlorophyll energy transfer efficiency over 90%. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

PubMed Central

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. PMID:29295818

Estimating the distance separating fluorescent protein FRET pairs

PubMed Central

van der Meer, B. Wieb; Blank, Paul S.

2014-01-01

Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10 nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs. PMID:23811334

HubAlign: an accurate and efficient method for global alignment of protein-protein interaction networks.

PubMed

Hashemifar, Somaye; Xu, Jinbo

2014-09-01

High-throughput experimental techniques have produced a large amount of protein-protein interaction (PPI) data. The study of PPI networks, such as comparative analysis, shall benefit the understanding of life process and diseases at the molecular level. One way of comparative analysis is to align PPI networks to identify conserved or species-specific subnetwork motifs. A few methods have been developed for global PPI network alignment, but it still remains challenging in terms of both accuracy and efficiency. This paper presents a novel global network alignment algorithm, denoted as HubAlign, that makes use of both network topology and sequence homology information, based upon the observation that topologically important proteins in a PPI network usually are much more conserved and thus, more likely to be aligned. HubAlign uses a minimum-degree heuristic algorithm to estimate the topological and functional importance of a protein from the global network topology information. Then HubAlign aligns topologically important proteins first and gradually extends the alignment to the whole network. Extensive tests indicate that HubAlign greatly outperforms several popular methods in terms of both accuracy and efficiency, especially in detecting functionally similar proteins. HubAlign is available freely for non-commercial purposes at http://ttic.uchicago.edu/∼hashemifar/software/HubAlign.zip. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

SBION: A Program for Analyses of Salt-Bridges from Multiple Structure Files.

PubMed

Gupta, Parth Sarthi Sen; Mondal, Sudipta; Mondal, Buddhadev; Islam, Rifat Nawaz Ul; Banerjee, Shyamashree; Bandyopadhyay, Amal K

2014-01-01

Salt-bridge and network salt-bridge are specific electrostatic interactions that contribute to the overall stability of proteins. In hierarchical protein folding model, these interactions play crucial role in nucleation process. The advent and growth of protein structure database and its availability in public domain made an urgent need for context dependent rapid analysis of salt-bridges. While these analyses on single protein is cumbersome and time-consuming, batch analyses need efficient software for rapid topological scan of a large number of protein for extracting details on (i) fraction of salt-bridge residues (acidic and basic). (ii) Chain specific intra-molecular salt-bridges, (iii) inter-molecular salt-bridges (protein-protein interactions) in all possible binary combinations (iv) network salt-bridges and (v) secondary structure distribution of salt-bridge residues. To the best of our knowledge, such efficient software is not available in public domain. At this juncture, we have developed a program i.e. SBION which can perform all the above mentioned computations for any number of protein with any number of chain at any given distance of ion-pair. It is highly efficient, fast, error-free and user friendly. Finally we would say that our SBION indeed possesses potential for applications in the field of structural and comparative bioinformatics studies. SBION is freely available for non-commercial/academic institutions on formal request to the corresponding author (akbanerjee@biotech.buruniv.ac.in).

Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis.

PubMed

Wang, Jia-Xing; Fang, Ge-Min; He, Yao; Qu, Da-Liang; Yu, Min; Hong, Zhang-Yong; Liu, Lei

2015-02-09

Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

PubMed

Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

2018-05-08

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Fast and Accurate Multivariate Gaussian Modeling of Protein Families: Predicting Residue Contacts and Protein-Interaction Partners

PubMed Central

Feinauer, Christoph; Procaccini, Andrea; Zecchina, Riccardo; Weigt, Martin; Pagnani, Andrea

2014-01-01

In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation) have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids), exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i) the prediction of residue-residue contacts in proteins, and (ii) the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code. PMID:24663061

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

PubMed

Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-01-01

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

PubMed

Leontovyc, Ivan; Habart, David; Loukotova, Sarka; Kosinova, Lucie; Kriz, Jan; Saudek, Frantisek; Koblas, Tomas

2017-01-01

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.

Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi

2014-03-28

Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with amore » Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.« less

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using Library MosSCI

PubMed Central

Kaymak, Ebru; Farley, Brian M.; Hay, Samantha A.; Li, Chihua; Ho, Samantha; Hartman, Daniel J.; Ryder, Sean P.

2016-01-01

Background In C. elegans, germline development and early embryogenesis rely on post-transcriptional regulation of maternally transcribed mRNAs. In many cases, the 3′UTR is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. Results In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RBPs that control the expression pattern of five different maternal mRNAs. Conclusions The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. PMID:27294288

Semiautomated Sample Preparation for Protein Stability and Formulation Screening via Buffer Exchange.

PubMed

Ying, William; Levons, Jaquan K; Carney, Andrea; Gandhi, Rajesh; Vydra, Vicky; Rubin, A Erik

2016-06-01

A novel semiautomated buffer exchange process workflow was developed to enable efficient early protein formulation screening. An antibody fragment protein, BMSdab, was used to demonstrate the workflow. The process afforded 60% to 80% cycle time and scientist time savings and significant material efficiencies. These efficiencies ultimately facilitated execution of this stability work earlier in the drug development process, allowing this tool to inform the developability of potential candidates for development from a formulation perspective. To overcome the key technical challenges, the protein solution was buffer-exchanged by centrifuge filtration into formulations for stability screening in a 96-well plate with an ultrafiltration membrane, leveraging automated liquid handling and acoustic volume measurements to allow several cycles of exchanges. The formulations were transferred into a vacuum manifold and sterile filtered into a rack holding 96 glass vials. The vials were sealed with a capmat of individual caps and placed in stability stations. Stability of the samples prepared by this process and by the standard process was demonstrated to be comparable. This process enabled screening a number of formulations of a protein at an early pharmaceutical development stage with a short sample preparation time. © 2015 Society for Laboratory Automation and Screening.

Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes

NASA Astrophysics Data System (ADS)

Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.

2016-06-01

A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

PubMed Central

Miller, Thomas F.

2017-01-01

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. PMID:28328943

The Efficiency of Different Salts to Screen Charge Interactions in Proteins: A Hofmeister Effect?

PubMed Central

Perez-Jimenez, Raul; Godoy-Ruiz, Raquel; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2004-01-01

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces. PMID:15041679

DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

PubMed

Kunig, Verena; Potowski, Marco; Gohla, Anne; Brunschweiger, Andreas

2018-06-27

DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.

2010-12-17

Research highlights: {yields} The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. {yields} Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. {yields} The intein splices with Lys in place of the highly conserved penultimate His. {yields} The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled tomore » ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.« less

Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier.

PubMed

Xu, Yongmei; Du, Yumin; Huang, Ronghua; Gao, Leping

2003-12-01

N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) is water-soluble derivative of chitosan (CS), synthesized by the reaction between glycidyl-trimethyl-ammonium chloride and CS. HTCC nanoparticles have been formed based on ionic gelation process of HTCC and sodium tripolyphosphate (TPP). Bovine serum albumin (BSA), as a model protein drug, was incorporated into the HTCC nanoparticles. HTCC nanoparticles were 110-180 nm in size, and their encapsulation efficiency was up to 90%. In vitro release studies showed a burst effect and a slow and continuous release followed. Encapsulation efficiency was obviously increased with increase of initial BSA concentration. Increasing TPP concentration from 0.5 to 0.7 mg/ml promoted encapsulation efficiency from 46.7% to 90%, and delayed release. As for modified HTCC nanoparticles, adding polyethylene glycol (PEG) or sodium alginate obviously decreased the burst effect of BSA from 42% to 18%. Encapsulation efficiency was significantly reduced from 47.6% to 2% with increase of PEG from 1.0 to 20.0 mg/ml. Encapsulation efficiency was increased from 14.5% to 25.4% with increase of alginate from 0.3 to 1.0 mg/ml.

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing

PubMed Central

Penn, Andrew C.; Balik, Ales; Greger, Ingo H.

2013-01-01

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site. PMID:23172291

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres▿

PubMed Central

Pike, Brietta L.; Heierhorst, Jörg

2007-01-01

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance. PMID:17636027

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

PubMed

Li, Haiou; Yao, Ruifeng; Ma, Sui; Hu, Shuai; Li, Suhua; Wang, Yupei; Yan, Chun; Xie, Daoxin; Yan, Jianbin

2017-11-01

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

PubMed Central

Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

2010-01-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

PubMed

Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

2017-03-24

In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants

PubMed Central

Wang, Ying; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.

2007-01-01

The most common cause of CF (cystic fibrosis) is the deletion of Phe508 (ΔF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with ΔF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of ΔF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of ΔF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of ΔF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of ΔF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ΔF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on ΔF508 CFTR maturation suggests that they directly interact at different sites of the protein. PMID:17535157

Biofunctionalized nanofibrous membranes as super separators of protein and enzyme from water.

PubMed

Homaeigohar, Shahin; Dai, Tianhe; Elbahri, Mady

2013-09-15

Here, we report development of a novel biofunctionalized nanofibrous membrane which, despite its macroporous structure, is able to separate even trace amounts (as low as 2mg/L) of biomolecules such as protein and enzyme from water with an optimum efficiency of ~90%. Such an extraordinary protein selectivity at this level of pollutant concentration for a nanofibrous membrane has never been reported. In the current study, poly(acrylonitrile-co-glycidyl methacrylate) (PANGMA) electrospun nanofibers are functionalized by a bovine serum albumin (BSA) protein. This membrane is extraordinarily successful in removal of BSA protein and Candida antarctica Lipase B (Cal-B) enzyme from a water based solution. Despite a negligible non-specific adsorption of both BSA and Cal-B to the PANGMA nanofibrous membrane (8%), the separation efficiency of the biofunctionalized membrane for BSA and Cal-B reaches to 88% and 81%, respectively. The optimum separation efficiency at a trace amount of protein models is due to the water-induced conformational change of the biofunctional agent. The conformational change not only exposes more functional groups available to catch the biomolecules but also leads to swelling of the nanofibers thereby a higher steric hindrance for the solutes. Besides the optimum selectivity, the biofunctionalized membranes are highly wettable thereby highly water permeable. Copyright © 2013 Elsevier Inc. All rights reserved.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

NASA Astrophysics Data System (ADS)

Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

2014-06-01

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

PubMed

Boissinot, Sylvaine; Erdinger, Monique; Monsion, Baptiste; Ziegler-Graff, Véronique; Brault, Véronique

2014-01-01

Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

Both Structural and Non-Structural Forms of the Readthrough Protein of Cucurbit aphid-borne yellows virus Are Essential for Efficient Systemic Infection of Plants

PubMed Central

Boissinot, Sylvaine; Erdinger, Monique; Monsion, Baptiste; Ziegler-Graff, Véronique; Brault, Véronique

2014-01-01

Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement. PMID:24691251

Actin-Binding Protein Requirement for Cortical Stability and Efficient Locomotion

NASA Astrophysics Data System (ADS)

Cunningham, C. Casey; Gorlin, Jed B.; Kwiatkowski, David J.; Hartwig, John H.; Janmey, Paul A.; Randolph Byers, H.; Stossel, Thomas P.

1992-01-01

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins.

PubMed

Kullolli, Majlinda; Rock, Dan A; Ma, Ji

2017-02-03

Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the N-terminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent liquid chromatgraphy-tandem mass spectroscopy. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTs-huFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2017-05-15

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice. © 2017. Published by The Company of Biologists Ltd.

Differential effects of high-fat diets varying in fatty acid composition on the efficiency of lean and fat tissue deposition during weight recovery after low food intake.

PubMed

Dulloo, A G; Mensi, N; Seydoux, J; Girardier, L

1995-02-01

The energetics of body weight recovery after low food intake was examined in the rat during refeeding for 2 weeks with isocaloric amounts of high-fat (HF) diets providing 50% of energy as either lard, coconut oil, olive oil, safflower oil, menhaden fish oil, or a mixture of all these fat types. The results indicate that for both body fat and protein, the efficiency of deposition was dependent on the dietary fat type. The most striking differences were found (1) between diets rich in n-3 and n-6 polyunsaturated fatty acids (PUFA), with the diet high in fish oil resulting in a greater body fat deposition and lower protein gain than the diet high in safflower oil; and (2) between diets rich in long-chain (LCT) and medium-chain triglycerides (MCT), with the diet high in lard resulting in a greater gain in both body fat and protein than the diet high in coconut oil. Furthermore, the diet high in olive oil (a monounsaturated fat) and the mixed-fat diet (containing all fat types) were found to be similar to the fish oil diet in that the efficiency of fat deposition was greater (and that of protein gain lower) than with the diet high in safflower oil. Neither the efficiency of fat gain nor that of protein gain were found to correlate with fasting plasma insulin, the insulin to glucose ratio, or plasma lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Improving the enzymolysis efficiency of potato protein by simultaneous dual-frequency energy-gathered ultrasound pretreatment: Thermodynamics and kinetics.

PubMed

Cheng, Yu; Liu, Yun; Wu, Juan; Ofori Donkor, Prince; Li, Ting; Ma, Haile

2017-07-01

The thermodynamics and kinetics of traditional and simultaneous dual frequency energy-gathered ultrasound (SDFU) assisted enzymolysis of potato protein were investigated to get the knowledge of the mechanisms on the SDFU's promoting efficiency during enzymolysis. The concentration of potato protein hydrolysate and parameters of thermodynamic and kinetic during traditional and SDFU assisted enzymolysis were determined. The results showed that potato protein hydrolysate concentration of SDFU assisted enzymolysis was higher than traditional enzymolysis at the hydrolysis time of 60min (p<0.05) whereas not significantly different at 120min (p>0.05). In some cases, SDFU assisted enzymolysis took less hydrolysis time than traditional enzymolysis when the similar conversion rates of potato protein were obtained. The thermodynamic papameters including the energy of activation (E a ), enthalpy of activation (△H), entropy of activation (△S) were reduced by ultrasound pretreatment while Gibbs free energy of activation (△G) increased little (1.6%). Also, kinetic papameters including Michaelis constant (K M ) and catalytic rate constant (k cat ) decreased by ultrasound pretreatment. On the contrary, reaction rate constants (k) of SDFU assisted enzymolysis were higher than that of traditional enzymolysis (p<0.05). It was indicated that the efficiency of SDFU assisted enzymolysis was higher than traditional enzymolysis in a limited time. The higher efficiency of SDFU assisted enzymolysis was related with the decrease of E a and K M by lowering the energy barrier between ground and active state and increasing affinity between substrate and enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of decreasing ideal protein levels on performance results and nitrogen efficiency of growing-finishing gilts.

PubMed

Millet, Sam; Aluwé, Marijke; De Paepe, Marc; De Brabander, Daniël L; Van Oeckel, Monique J

2010-02-01

This study examined the effect of decreasing ideal protein concentrations on performance and nutrient efficiency. The experimental diets contained 100%, 90%, 80% or 70% of the ideal dietary protein level (Diet 100%, 90%, 80% and 70%, respectively) estimated in previous experiments with pigs of the same genetic background. The four different treatments were divided among 16 pens of six pigs each. The average initial and final body weight were 20.8 +/- 1.1 and 107 +/- 3 kg, respectively. Three-phase feeding was applied (BW 20-40 kg, 40-70 kg and 70-110 kg). The dietary ileal digestible (ID) methionine + cystine, threonine, tryptophan, isoleucine, valine, and leucine contents expressed as percent of ID lysine were 63, 72, 22, 60, 68, and > 100%, respectively. The lysine to protein ratio was kept constant at 6.8%. Between 21 and 106 kg BW the best performance was achieved on Diet 90%. Diet 70% led to significantly worse results. Although lean meat percentage did not differ, protein content of the carcass was lower on Diet 70% than on Diets 90% and 100%. Decreased protein concentrations increased crude protein efficiency and consequently decreased nitrogen excretion most at the 80% level. If protein varies together with digestible amino acid content, it can be concluded that Diet 80% may be the best choice for the environment and for profitability. If protein content stays at a fixed level, Diet 90% may be the safest choice.

Increase in local protein concentration by field-inversion gel electrophoresis.

PubMed

Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

2007-09-26

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Body composition, protein and energy efficiencies, and requirements for growth of F1 Boer × Saanen goat kids.

PubMed

Teixeira, I A M A; Fernandes, M H M R; Filho, J M Pereira; Canesin, R C; Gomes, R A; Resende, K T

2017-05-01

We conducted a study in which body composition, energy and protein requirements, and efficiency of MP and ME were determined in F1 Boer × Saanen goat kids of 5 to 25 kg BW by using the comparative slaughter technique. Two experiments were performed: Exp. 1 estimated the maintenance requirements of kids from 15 to 25 kg BW, and Exp. 2 estimated the gain requirements of kids from 5 to 25 kg BW. In Exp. 1, 28 intact male F1 Boer × Saanen goat kids were utilized, with 7 kids slaughtered (BW of 15.0 ± 0.35 kg) at the onset for estimation of initial body composition and the remaining 21 kids assigned to a randomized block design. Within each block, kids were subjected to 3 levels of feed intake treatments (ad libitum [100%] or restricted to 70% or 40% ad libitum). All kids in each block were slaughtered when the animals fed ad libitum reached 25 kg BW. The NE, ME for maintenance, and partial efficiency of use of ME for NE were 321.6 kJ/kg BW, 525.9 kJ/kg BW, and 0.61, respectively. The net protein and MP for maintenance were 2.43 g/kg of BW and 4.41 g/kg of BW, respectively; thus, the estimated partial efficiency of MP for maintenance was 0.55. In Exp. 2, 32 intact male F1 Boer × Saanen goat kids were distributed in a completely randomized design and slaughtered at 5.6 ± 0.85 kg BW ( = 6), 10.0 ± 0.35 kg BW ( = 6), 15.3 ± 0.52 kg BW ( = 7), 20.4 ± 0.66 kg BW ( = 6), and 25 ± 0.46 kg BW ( = 7). Body composition was then fitted to allometric equations. Body fat composition increased from 37 to 114 g/kg empty BW (EBW; < 0.001), and body protein composition decreased by 10% (from 203.2 to 180.6 g/kg EBW; < 0.001) when kids grew from 5 to 25 kg BW. The NE increased by approximately 60% (from 7.2 to 11.5 MJ/kg of empty BW gain [EWG]; < 0.001), and the net protein for gain decreased by 10% (from 186 to 166 g/kg of EWG; < 0.001). The partial efficiency of the utilization of ME to NE for growth was 0.32 ( < 0.0001), and the partial efficiencies of the utilization of ME for the synthesis of protein and fat were 0.19 and 0.59 ( < 0.011), respectively. These results demonstrate that the protein and energy maintenance requirements in young crossbred goat kids are greater than values reported previously in feeding system studies. In addition, their requirements for gain depend on body composition and are driven by efficiencies of deposition.

Protein engineering in designing tailored enzymes and microorganisms for biofuels production

PubMed Central

Wen, Fei; Nair, Nikhil U; Zhao, Huimin

2009-01-01

Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

Short communication: Feed utilization and its associations with fertility and productive life in 11 commercial Pennsylvania tie-stall herds.

PubMed

Vallimont, J E; Dechow, C D; Daubert, J M; Dekleva, M W; Blum, J W; Liu, W; Varga, G A; Heinrichs, A J; Baumrucker, C R

2013-02-01

The objectives of this study were to quantify the relationships of various definitions of feed utilization with both fertility and productive life. Intake and body measurement data were collected monthly on 970 cows in 11 tie-stall herds for 6 consecutive months. Measures of feed utilization for this study were dry matter intake (DMI), dry matter intake efficiency (DME, defined as 305-d fat-corrected milk/305-d DMI), DME with intake adjusted for maintenance requirements (DMEM), crude protein efficiency (defined as 305-d protein yield/305-d crude protein intake), and 2 definitions of residual feed intake (RFI). The first, RFI(reg), was calculated by regressing daily DMI on daily milk, fat, and protein yields, body weight (BW), daily body condition score (BCS) gain or loss, the interaction between BW and BCS gain or loss, and days in milk. The second, RFI(NRC), was estimated by subtracting 305-d DMI predicted according to their fat-corrected milk and BW from actual 305-d DMI. Data were analyzed with 8-trait animal models and included one measure of feed utilization and milk, fat, and protein yields, BW, BCS, days open (DO), and productive life (PL). The genetic correlation between DME and DO was 0.53 (± 0.19) and that between DME and PL was 0.66 (± 0.10). These results show that cows who had higher feed efficiency had greater DO (undesirable) and greater PL (desirable). Results were similar for the genetic correlation between DO and crude protein efficiency (0.42). Productive life had genetic correlations of -0.22 with BW and -0.48 with BCS, suggesting that larger, fatter cows in this study had shorter PL. Correlations between estimated breeding values for feed utilization and official sire genetic evaluations for fertility were in agreement with the results from the multiple-trait models. Selection programs intended to enhance feed efficiency should factor relationships with functional traits to avoid unfavorable effects on cow fertility. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Efficiency of Adaptive Temperature-Based Replica Exchange for Sampling Large-Scale Protein Conformational Transitions.

PubMed

Zhang, Weihong; Chen, Jianhan

2013-06-11

Temperature-based replica exchange (RE) is now considered a principal technique for enhanced sampling of protein conformations. It is also recognized that existence of sharp cooperative transitions (such as protein folding/unfolding) can lead to temperature exchange bottlenecks and significantly reduce the sampling efficiency. Here, we revisit two adaptive temperature-based RE protocols, namely, exchange equalization (EE) and current maximization (CM), that were previously examined using atomistic simulations (Lee and Olson, J. Chem. Physics2011, 134, 24111). Both protocols aim to overcome exchange bottlenecks by adaptively adjusting the simulation temperatures, either to achieve uniform exchange rates (in EE) or to maximize temperature diffusion (CM). By designing a realistic yet computationally tractable coarse-grained protein model, one can sample many reversible folding/unfolding transitions using conventional constant temperature molecular dynamics (MD), standard REMD, EE-REMD, and CM-REMD. This allows rigorous evaluation of the sampling efficiency, by directly comparing the rates of folding/unfolding transitions and convergence of various thermodynamic properties of interest. The results demonstrate that both EE and CM can indeed enhance temperature diffusion compared to standard RE, by ∼3- and over 10-fold, respectively. Surprisingly, the rates of reversible folding/unfolding transitions are similar in all three RE protocols. The convergence rates of several key thermodynamic properties, including the folding stability and various 1D and 2D free energy surfaces, are also similar. Therefore, the efficiency of RE protocols does not appear to be limited by temperature diffusion, but by the inherent rates of spontaneous large-scale conformational rearrangements. This is particularly true considering that virtually all RE simulations of proteins in practice involve exchange attempt frequencies (∼ps(-1)) that are several orders of magnitude faster than the slowest protein motions (∼μs(-1)). Our results also suggest that the efficiency of RE will not likely be improved by other protocols that aim to accelerate exchange or temperature diffusion. Instead, protocols with some types of guided tempering will likely be necessary to drive faster large-scale conformational transitions.

Quantifying ruminal nitrogen metabolism using the omasal sampling technique in cattle--a meta-analysis.

PubMed

Broderick, G A; Huhtanen, P; Ahvenjärvi, S; Reynal, S M; Shingfield, K J

2010-07-01

Mixed model analysis of data from 32 studies (122 diets) was used to evaluate the precision and accuracy of the omasal sampling technique for quantifying ruminal-N metabolism and to assess the relationships between nonammonia-N flow at the omasal canal and milk protein yield. Data were derived from experiments in cattle fed North American diets (n=36) based on alfalfa silage, corn silage, and corn grain and Northern European diets (n=86) composed of grass silage and barley-based concentrates. In all studies, digesta flow was quantified using a triple-marker approach. Linear regressions were used to predict microbial-N flow to the omasum from intake of dry matter (DM), organic matter (OM), or total digestible nutrients. Efficiency of microbial-N synthesis increased with DM intake and there were trends for increased efficiency with elevated dietary concentrations of crude protein (CP) and rumen-degraded protein (RDP) but these effects were small. Regression of omasal rumen-undegraded protein (RUP) flow on CP intake indicated that an average 32% of dietary CP escaped and 68% was degraded in the rumen. The slope from regression of observed omasal flows of RUP on flows predicted by the National Research Council (2001) model indicated that NRC predicted greater RUP supply. Measured microbial-N flow was, on average, 26% greater than that predicted by the NRC model. Zero ruminal N-balance (omasal CP flow=CP intake) was obtained at dietary CP and RDP concentrations of 147 and 106 g/kg of DM, corresponding to ruminal ammonia-N and milk urea N concentrations of 7.1 and 8.3mg/100mL, respectively. Milk protein yield was positively related to the efficiency of microbial-N synthesis and measured RUP concentration. Improved efficiency of microbial-N synthesis and reduced ruminal CP degradability were positively associated with efficiency of capture of dietary N as milk N. In conclusion, the results of this study indicate that the omasal sampling technique yields valuable estimates of RDP, RUP, and ruminal microbial protein supply in cattle. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Application of Enhanced Sampling Monte Carlo Methods for High-Resolution Protein-Protein Docking in Rosetta

PubMed Central

Zhang, Zhe; Schindler, Christina E. M.; Lange, Oliver F.; Zacharias, Martin

2015-01-01

The high-resolution refinement of docked protein-protein complexes can provide valuable structural and mechanistic insight into protein complex formation complementing experiment. Monte Carlo (MC) based approaches are frequently applied to sample putative interaction geometries of proteins including also possible conformational changes of the binding partners. In order to explore efficiency improvements of the MC sampling, several enhanced sampling techniques, including temperature or Hamiltonian replica exchange and well-tempered ensemble approaches, have been combined with the MC method and were evaluated on 20 protein complexes using unbound partner structures. The well-tempered ensemble method combined with a 2-dimensional temperature and Hamiltonian replica exchange scheme (WTE-H-REMC) was identified as the most efficient search strategy. Comparison with prolonged MC searches indicates that the WTE-H-REMC approach requires approximately 5 times fewer MC steps to identify near native docking geometries compared to conventional MC searches. PMID:26053419

Targeted gene disruption in Koji mold Aspergillus oryzae.

PubMed

Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2011-01-01

Filamentous fungi have received attentions as hosts for heterologous protein production because of their high secretion capability and eukaryotic post-translational modifications. One of the safest hosts for heterologous protein production is Koji mold Aspergillus oryzae since it has been used in the production of Japanese fermented foods for over 1,000 years. The production levels of proteins from higher eukaryotes are much lower than those of homologous (fungal) proteins. Bottlenecks in the heterologous protein production are suggested to be proteolytic degradation of the produced protein in the medium and the secretory pathway. For construction of excellent host strains, many genes causing the bottlenecks should be disrupted rapidly and efficiently. We developed a marker recycling system with the highly efficient gene-targeting background in A. oryzae. By employing this technique, we performed multiple gene disruption of the ten protease genes. The decuple protease gene disruptant showed fourfold production level of a heterologous protein compared with the wild-type strain.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Reciprocal Efficiency of RNQ1 and Polyglutamine Detoxification in the Cytosol and Nucleus

PubMed Central

Douglas, Peter M.; Summers, Daniel W.; Ren, Hong-Yu

2009-01-01

Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon the intracellular presence of [RNQ+] prions. Increasing heat-shock protein 40 chaperone activity before Rnq1-GFP expression, shifted Rnq1-GFP aggregation from the cytosol to the nucleus. Assembly of Rnq1-GFP into benign amyloid-like aggregates was more efficient in the nucleus than cytosol and nuclear accumulation of Rnq1-GFP correlated with reduced toxicity. [RNQ+] prions were found to form stable complexes with Htt-103Q, and nuclear Rnq1-GFP aggregates were capable of sequestering Htt-103Q in the nucleus. On accumulation in the nucleus, conversion of Htt-103Q into SDS-resistant aggregates was dramatically reduced and Htt-103Q toxicity was exacerbated. Alterations in activity of molecular chaperones, the localization of intracellular interaction partners, or both can impact the cellular location of disease proteins. This, in turn, impacts proteotoxicity because the assembly of proteins to a benign state occurs with different efficiencies in the cytosol and nucleus. PMID:19656852

Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.

PubMed

Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum

2017-01-01

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cell-penetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines.

Programmable In Vitro Coencapsidation of Guest Proteins for Intracellular Delivery by Virus-like Particles.

PubMed

Dashti, Noor H; Abidin, Rufika S; Sainsbury, Frank

2018-05-22

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages are being developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of bionanotechnology platforms that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of murine polyomavirus (MPyV) that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer, we demonstrate the flexibility in this system to support coencapsidation of multiple proteins. Complementing these ensemble measurements with single-particle analysis by super-resolution microscopy shows that the stochastic nature of coencapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable coencapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Bio-Photoelectrochemical Solar Cells Incorporating Reaction Center and Reaction Center Plus Light Harvesting Complexes

NASA Astrophysics Data System (ADS)

Yaghoubi, Houman

Harvesting solar energy can potentially be a promising solution to the energy crisis now and in the future. However, material and processing costs continue to be the most important limitations for the commercial devices. A key solution to these problems might lie within the development of bio-hybrid solar cells that seeks to mimic photosynthesis to harvest solar energy and to take advantage of the low material costs, negative carbon footprint, and material abundance. The bio-photoelectrochemical cell technologies exploit biomimetic means of energy conversion by utilizing plant-derived photosystems which can be inexpensive and ultimately the most sustainable alternative. Plants and photosynthetic bacteria harvest light, through special proteins called reaction centers (RCs), with high efficiency and convert it into electrochemical energy. In theory, photosynthetic RCs can be used in a device to harvest solar energy and generate 1.1 V open circuit voltage and ~1 mA cm-2 short circuit photocurrent. Considering the nearly perfect quantum yield of photo-induced charge separation, efficiency of a protein-based solar cell might exceed 20%. In practice, the efficiency of fabricated devices has been limited mainly due to the challenges in the electron transfer between the protein complex and the device electrodes as well as limited light absorption. The overarching goal of this work is to increase the power conversion efficiency in protein-based solar cells by addressing those issues (i.e. electron transfer and light absorption). This work presents several approaches to increase the charge transfer rate between the photosynthetic RC and underlying electrode as well as increasing the light absorption to eventually enhance the external quantum efficiency (EQE) of bio-hybrid solar cells. The first approach is to decrease the electron transfer distance between one of the redox active sites in the RC and the underlying electrode by direct attachment of the of protein complex onto Au electrodes via surface exposed cysteine residues. This resulted in photocurrent densities as large as ~600 nA cm-2 while still the incident photon to generated electron quantum efficiency was as low as %3 x 10-4. 2- The second approach is to immobilize wild type RCs of Rhodobacter sphaeroides on the surface of a Au underlying electrode using self-assembled monolayers of carboxylic acid terminated oligomers and cytochrome c charge mediating layers, with a preferential orientation from the primary electron donor site. This approach resulted in EQE of up to 0.06%, which showed 200 times efficiency improvement comparing to the first approach. In the third approach, instead of isolated protein complexes, RCs plus light harvesting (LH) complexes were employed for a better photon absorption. Direct attachment of RC-LH1 complexes on Au working electrodes, resulted in 0.21% EQE which showed 3.5 times efficiency improvement over the second approach (700 times higher than the first approach). The main impact of this work is the harnessing of biological RCs for efficient energy harvesting in man-made structures. Specifically, the results in this work will advance the application of RCs in devices for energy harvesting and will enable a better understanding of bio and nanomaterial interfaces, thereby advancing the application of biological materials in electronic devices. At the end, this work offers general guidelines that can serve to improve the performance of bio-hybrid solar cells.

The efficiency of cellular energy transduction and its implications for obesity.

PubMed

Harper, Mary-Ellen; Green, Katherine; Brand, Martin D

2008-01-01

We assess the existence, mechanism, and functions of less-than-maximal coupling efficiency of mitochondrial oxidative phosphorylation and its potential as a target for future antiobesity interventions. Coupling efficiency is the proportion of oxygen consumption used to make adenosine triphosphate (ATP) and do useful work. High coupling efficiency may lead to fat deposition; low coupling efficiency to a decrease in fat stores. We review obligatory and facultative energy expenditure and the role of a futile cycle of proton pumping and proton leak across the mitochondrial inner membrane in dissipating energy. Basal proton conductance is catalyzed primarily by the adenine nucleotide translocase but can be mimicked by chemical uncouplers. Inducible proton conductance is catalyzed by specific uncoupling proteins. We discuss the opportunities and pitfalls of targeting these processes as a treatment for obesity by decreasing coupling efficiency and increasing energy expenditure, either directly or through central mechanisms of energy homeostasis.

Improved efficiency of nanoneedle insertion by modification with a cell-puncturing protein

NASA Astrophysics Data System (ADS)

Ryu, Seunghwan; Matsumoto, Yuta; Matsumoto, Takahiro; Ueno, Takafumi; Silberberg, Yaron R.; Nakamura, Chikashi

2018-03-01

An atomic force microscope (AFM) probe etched into an ultra-sharp cylindrical shape (a nanoneedle) can be inserted into a living cell and mechanical responses of the insertion process are represented as force-distance curves using AFM. A probe-molecule-functionalized nanoneedle can be used to detect intracellular molecules of interest in situ. The insertion efficiencies of nanoneedles vary among cell types due to the cortex structures of cells, and some cell types, such as mouse fibroblast Balb/3T3 cells, show extremely low efficacy of insertion. We addressed this issue by using a cell membrane puncturing protein from bacteriophage T4 (gp5), a needle-like protein that spontaneously penetrates through the cell membrane. Gp5 was immobilized onto a nanoneedle surface. The insertion efficiency of the functionalized nanoneedle increased by over 15% compared to the non-functionalized control. Gp5-modification is a versatile approach in cell manipulation techniques for the insertion of other types of nanostructures into cells.

Snf1-related kinase improves cardiac mitochondrial efficiency and decreases mitochondrial uncoupling

PubMed Central

Rines, Amy K.; Chang, Hsiang-Chun; Wu, Rongxue; Sato, Tatsuya; Khechaduri, Arineh; Kouzu, Hidemichi; Shapiro, Jason; Shang, Meng; Burke, Michael A.; Abdelwahid, Eltyeb; Jiang, Xinghang; Chen, Chunlei; Rawlings, Tenley A.; Lopaschuk, Gary D.; Schumacker, Paul T.; Abel, E. Dale; Ardehali, Hossein

2017-01-01

Ischaemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in tissue death. Here, we demonstrate that the AMP-activated protein kinase (AMPK)-related protein Snf1-related kinase (SNRK) decreases cardiac metabolic substrate usage and mitochondrial uncoupling, and protects against ischaemia/reperfusion. Hearts from transgenic mice overexpressing SNRK have decreased glucose and palmitate metabolism and oxygen consumption, but maintained power and function. They also exhibit decreased uncoupling protein 3 (UCP3) and mitochondrial uncoupling. Conversely, Snrk knockout mouse hearts have increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreases mitochondrial efficiency, which is abolished with UCP3 knockdown. We show that Tribbles homologue 3 (Trib3) binds to SNRK, and downregulates UCP3 through PPARα. Finally, SNRK is increased in cardiomyopathy patients, and SNRK reduces infarct size after ischaemia/reperfusion. SNRK also decreases cardiac cell death in a UCP3-dependent manner. Our results suggest that SNRK improves cardiac mitochondrial efficiency and ischaemic protection. PMID:28117339

Identification of Immunogenic Hot Spots within Plum Pox Potyvirus Capsid Protein for Efficient Antigen Presentation

PubMed Central

Fernández-Fernández, M. Rosario; Martínez-Torrecuadrada, Jorge L.; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

2002-01-01

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-γ, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence. PMID:12438590

Flotation removal of the microalga Nannochloropsis sp. using Moringa protein-oil emulsion: A novel green approach.

PubMed

Kandasamy, Ganesan; Shaleh, Sitti Raehanah Muhamad

2018-01-01

A new approach to recover microalgae from aqueous medium using a bio-flotation method is reported. The method involves utilizing a Moringa protein extract - oil emulsion (MPOE) for flotation removal of Nannochloropsis sp. The effect of various factors has been assessed using this method, including operating parameters such as pH, MPOE dose, algae concentration and mixing time. A maximum flotation efficiency of 86.5% was achieved without changing the pH condition of algal medium. Moreover, zeta potential analysis showed a marked difference in the zeta potential values when increase the MPOE dose concentration. An optimum condition of MPOE dosage of 50ml/L, pH 8, mixing time 4min, and a flotation efficiency of greater than 86% was accomplished. The morphology of algal flocs produced by protein-oil emulsion flocculant were characterized by microscopy. This flotation method is not only simple, but also an efficient method for harvesting microalgae from culture medium. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial growth laws reflect the evolutionary importance of energy efficiency.

PubMed

Maitra, Arijit; Dill, Ken A

2015-01-13

We are interested in the balance of energy and protein synthesis in bacterial growth. How has evolution optimized this balance? We describe an analytical model that leverages extensive literature data on growth laws to infer the underlying fitness landscape and to draw inferences about what evolution has optimized in Escherichia coli. Is E. coli optimized for growth speed, energy efficiency, or some other property? Experimental data show that at its replication speed limit, E. coli produces about four mass equivalents of nonribosomal proteins for every mass equivalent of ribosomes. This ratio can be explained if the cell's fitness function is the the energy efficiency of cells under fast growth conditions, indicating a tradeoff between the high energy costs of ribosomes under fast growth and the high energy costs of turning over nonribosomal proteins under slow growth. This model gives insight into some of the complex nonlinear relationships between energy utilization and ribosomal and nonribosomal production as a function of cell growth conditions.

Removal of microbial multi-species biofilms from the paper industry by enzymatic treatments.

PubMed

Marcato-Romain, C E; Pechaud, Y; Paul, E; Girbal-Neuhauser, E; Dossat-Létisse, V

2012-01-01

This study aimed to characterize biofilms from the paper industry and evaluate the effectiveness of enzymatic treatments in reducing them. The extracellular polymeric substances (EPS) extracted from six industrial biofilms were studied. EPS were mainly proteins, the protein to polysaccharide ratio ranging from 1.3 to 8.6 depending on where the sampling point was situated in the paper making process. Eight hydrolytic enzymes were screened on a 24-h multi-species biofilm. The enzymes were tested at various concentrations and contact durations. Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while proteases were more efficient: after treatment for 24 h with pepsin, Alcalase® or Savinase®, the removal exceeded 80%. Savinase® appeared to be the most adequate for industrial conditions and was tested on an industrial biofilm sample. This enzyme led to a significant release of proteins from the EPS matrix, indicating its potential efficiency on an industrial scale.

Lignin-blocking treatment of biomass and uses thereof

DOEpatents

Yang, Bin [Hanover, NH; Wyman, Charles E [Norwich, VT

2009-10-20

Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion. Cellulase efficiencies are improved by the protein or polypeptide treatment. The treatment may be used in combination with steam explosion and acid prehydrolysis techniques. Hydrolysis yields from lignin containing biomass are enhanced 5-20%, and enzyme utilization is increased from 10% to 50%. Thus, a more efficient and economical method of processing lignin containing biomass materials utilizes a polypeptide/protein treatment step that effectively blocks lignin binding of cellulase.

A generative, probabilistic model of local protein structure.

PubMed

Boomsma, Wouter; Mardia, Kanti V; Taylor, Charles C; Ferkinghoff-Borg, Jesper; Krogh, Anders; Hamelryck, Thomas

2008-07-01

Despite significant progress in recent years, protein structure prediction maintains its status as one of the prime unsolved problems in computational biology. One of the key remaining challenges is an efficient probabilistic exploration of the structural space that correctly reflects the relative conformational stabilities. Here, we present a fully probabilistic, continuous model of local protein structure in atomic detail. The generative model makes efficient conformational sampling possible and provides a framework for the rigorous analysis of local sequence-structure correlations in the native state. Our method represents a significant theoretical and practical improvement over the widely used fragment assembly technique by avoiding the drawbacks associated with a discrete and nonprobabilistic approach.

A highly efficient dual-diazonium reagent for protein crosslinking and construction of a virus-based gel.

PubMed

Ma, Dejun; Zhang, Jie; Zhang, Changyu; Men, Yuwen; Sun, Hongyan; Li, Lu-Yuan; Yi, Long; Xi, Zhen

2018-05-09

A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-Efficiency Promoter-Dependent Transduction by Adeno-Associated Virus Type 6 Vectors in Mouse Lung

PubMed Central

HALBERT, CHRISTINE L.; LAM, SIU-LING; MILLER, A. DUSTY

2014-01-01

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, β-actin promoter and splice donor, and a β-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, highly efficient transduction can be achieved. PMID:17430088

Structure optimisation by thermal cycling for the hydrophobic-polar lattice model of protein folding

NASA Astrophysics Data System (ADS)

Günther, Florian; Möbius, Arnulf; Schreiber, Michael

2017-03-01

The function of a protein depends strongly on its spatial structure. Therefore the transition from an unfolded stage to the functional fold is one of the most important problems in computational molecular biology. Since the corresponding free energy landscapes exhibit huge numbers of local minima, the search for the lowest-energy configurations is very demanding. Because of that, efficient heuristic algorithms are of high value. In the present work, we investigate whether and how the thermal cycling (TC) approach can be applied to the hydrophobic-polar (HP) lattice model of protein folding. Evaluating the efficiency of TC for a set of two- and three-dimensional examples, we compare the performance of this strategy with that of multi-start local search (MSLS) procedures and that of simulated annealing (SA). For this aim, we incorporated several simple but rather efficient modifications into the standard procedures: in particular, a strong improvement was achieved by also allowing energy conserving state modifications. Furthermore, the consideration of ensembles instead of single samples was found to greatly improve the efficiency of TC. In the framework of different benchmarks, for all considered HP sequences, we found TC to be far superior to SA, and to be faster than Wang-Landau sampling.

Opportunities for improving milk production efficiency in dairy cattle

USDA-ARS?s Scientific Manuscript database

Increasing feed costs and the desire to improve environmental stewardship have stimulated interest in improving feed efficiency of livestock, including that of U.S. dairy herds. For instance, USDA cost projections for corn and soybean meal suggest a 20% increase over 2010 pricing for a 16% protein ...

Physical-chemical features of non-detergent sulfobetaines active as protein-folding helpers.

PubMed

Expert-Bezançon, Nicole; Rabilloud, Thierry; Vuillard, Laurent; Goldberg, Michel E

2003-01-01

Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.

Highly Disordered Amyloid-β Monomer Probed by Single-Molecule FRET and MD Simulation.

PubMed

Meng, Fanjie; Bellaiche, Mathias M J; Kim, Jae-Yeol; Zerze, Gül H; Best, Robert B; Chung, Hoi Sung

2018-02-27

Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies. Published by Elsevier Inc.

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

PubMed Central

van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

Structural alignment of protein descriptors - a combinatorial model.

PubMed

Antczak, Maciej; Kasprzak, Marta; Lukasiak, Piotr; Blazewicz, Jacek

2016-09-17

Structural alignment of proteins is one of the most challenging problems in molecular biology. The tertiary structure of a protein strictly correlates with its function and computationally predicted structures are nowadays a main premise for understanding the latter. However, computationally derived 3D models often exhibit deviations from the native structure. A way to confirm a model is a comparison with other structures. The structural alignment of a pair of proteins can be defined with the use of a concept of protein descriptors. The protein descriptors are local substructures of protein molecules, which allow us to divide the original problem into a set of subproblems and, consequently, to propose a more efficient algorithmic solution. In the literature, one can find many applications of the descriptors concept that prove its usefulness for insight into protein 3D structures, but the proposed approaches are presented rather from the biological perspective than from the computational or algorithmic point of view. Efficient algorithms for identification and structural comparison of descriptors can become crucial components of methods for structural quality assessment as well as tertiary structure prediction. In this paper, we propose a new combinatorial model and new polynomial-time algorithms for the structural alignment of descriptors. The model is based on the maximum-size assignment problem, which we define here and prove that it can be solved in polynomial time. We demonstrate suitability of this approach by comparison with an exact backtracking algorithm. Besides a simplification coming from the combinatorial modeling, both on the conceptual and complexity level, we gain with this approach high quality of obtained results, in terms of 3D alignment accuracy and processing efficiency. All the proposed algorithms were developed and integrated in a computationally efficient tool descs-standalone, which allows the user to identify and structurally compare descriptors of biological molecules, such as proteins and RNAs. Both PDB (Protein Data Bank) and mmCIF (macromolecular Crystallographic Information File) formats are supported. The proposed tool is available as an open source project stored on GitHub ( https://github.com/mantczak/descs-standalone ).

Effects of Meat Cooking, and of Ingested Amount, on Protein Digestion Speed and Entry of Residual Proteins into the Colon: A Study in Minipigs

PubMed Central

Bax, Marie-Laure; Buffière, Caroline; Hafnaoui, Noureddine; Gaudichon, Claire; Savary-Auzeloux, Isabelle; Dardevet, Dominique; Santé-Lhoutellier, Véronique; Rémond, Didier

2013-01-01

The speed of protein digestion impacts on postprandial protein anabolism. After exercise or in the elderly, fast proteins stimulate protein synthesis more efficiently than slow proteins. It has been shown that meat might be a source of fast proteins. However, cooking temperature, acting on the macrostructure and microstructure of the meat could affect both the speed, and efficiency, of protein digestion. This study aims to evaluate, in vivo, the effect of meat cooking on digestion parameters, in the context of a complete meal. Six minipigs fitted with an ileal cannula and an arterial catheter were used. In order to measure the true ileal digestibility, tested meat was obtained from a calf, the muscle proteins of which were intrinsically labelled with 15N-amino acids. Three cooking temperatures (60, 75 and 95°C; core temperature for 30 min), and three levels of intake (1, 1.45, and 1.90 g protein/kg body weight) were tested. Following meat ingestion, ileal digesta and arterial blood were collected over a 9-h period. The speed of digestion, evaluated from the kinetics of amino acid appearance in blood within the first 3 h, was greater for the cooking temperature of 75°C, than for 60 or 95°C. The true ileal digestibility, which averaged 95%, was not affected by cooking temperature or by the level of meat intake. The amino acid composition of the digesta flowing at the ileum was not affected by cooking temperature. These results show that cooking temperature can modulate the speed of meat protein digestion, without affecting the efficiency of the small intestinal digestion, and consequently the entry of meat protein residues into the colon. PMID:23593443

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

PubMed

Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

2008-05-16

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.

PubMed

Deb-Choudhury, Santanu; Plowman, Jeffrey E; Harland, Duane P

2016-01-01

The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Quinoa seed quality response to sodium chloride and sodium sulfate salinity

USDA-ARS?s Scientific Manuscript database

Quinoa (Chenopodium Quinoa Willd.) is a pseudocereal which has high protein content, but also provides high quality protein. The essential amino acids are balanced; protein efficiency ratio and true protein digestibility are comparable to those of casein, which considered as a good protein resource....

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Weihong; Wu Jianqing; Zhong Li

2006-09-30

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency ofmore » conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

Impact of microbial efficiency to predict MP supply when estimating protein requirements of growing beef cattle from performance.

PubMed

Watson, A K; Klopfenstein, T J; Erickson, G E; MacDonald, J C; Wilkerson, V A

2017-07-01

Data from 16 trials were compiled to calculate microbial CP (MCP) production and MP requirements of growing cattle on high-forage diets. All cattle were individually fed diets with 28% to 72% corn cobs in addition to either alfalfa, corn silage, or sorghum silage at 18% to 60% of the diet (DM basis). The remainder of the diet consisted of protein supplement. Source of protein within the supplement varied and included urea, blood meal, corn gluten meal, dry distillers grains, feather meal, meat and bone meal, poultry by-product meal, soybean meal, and wet distillers grains. All trials included a urea-only treatment. Intake of all cattle within an experiment was held constant, as a percentage of BW, established by the urea-supplemented group. In each trial the base diet (forage and urea supplement) was MP deficient. Treatments consisted of increasing amounts of test protein replacing the urea supplement. As protein in the diet increased, ADG plateaued. Among experiments, ADG ranged from 0.11 to 0.73 kg. Three methods of calculating microbial efficiency were used to determine MP supply. Gain was then regressed against calculated MP supply to determine MP requirement for maintenance and gain. Method 1 (based on a constant 13% microbial efficiency as used by the beef NRC model) predicted an MP maintenance requirement of 3.8 g/kg BW and 385 g MP/kg gain. Method 2 calculated microbial efficiency using low-quality forage diets and predicted MP requirements of 3.2 g/kg BW for maintenance and 448 g/kg for gain. Method 3 (based on an equation predicting MCP yield from TDN intake, proposed by the Beef Cattle Nutrient Requirements Model [BCNRM]) predicted MP requirements of 3.1 g/kg BW for maintenance and 342 g/kg for gain. The factorial method of calculating MP maintenance requirements accounts for scurf, endogenous urinary, and metabolic fecal protein losses and averaged 4.2 g/kg BW. Cattle performance data demonstrate formulating diets to meet the beef NRC model recommended MP maintenance requirement (3.8 g/kg S) works well when using 13% microbial efficiency. Therefore, a change in how microbial efficiency is calculated necessitates a change in the proposed MP maintenance requirement to not oversupply or undersupply RUP. Using the 2016 BCNRM to predict MCP production and formulate diets to meet MP requirements also requires changing the MP maintenance requirement to 3.1 g/kg BW.

Degradation properties of protein and carbohydrate during sludge anaerobic digestion.

PubMed

Yang, Guang; Zhang, Panyue; Zhang, Guangming; Wang, Yuanyuan; Yang, Anqi

2015-09-01

Degradation of protein and carbohydrate is vital for sludge anaerobic digestion performance. However, few studies focused on degradation properties of protein and carbohydrate. This study investigated detailed degradation properties of sludge protein and carbohydrate in order to gain insight into organics removal during anaerobic digestion. Results showed that carbohydrate was more efficiently degraded than protein and was degraded prior to protein. The final removal efficiencies of carbohydrate and protein were 49.7% and 32.2%, respectively. The first 3 days were a lag phase for protein degradation since rapid carbohydrate degradation in this phase led to repression of protease formation. Kinetics results showed that, after initial lag phase, protein degradation followed the first-order kinetic with rate constants of 0.0197 and 0.0018 d(-1) during later rapid degradation phase and slow degradation phase, respectively. Carbohydrate degradation also followed the first-order kinetics with a rate constant of 0.007 d(-1) after initial quick degradation phase. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

PubMed

Błachnio, Karina

2010-01-01

Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.

PubMed

Tian, Ji; Pei, Haixia; Zhang, Shuai; Chen, Jiwei; Chen, Wen; Yang, Ruoyun; Meng, Yonglu; You, Jie; Gao, Junping; Ma, Nan

2014-01-01

Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.

TRV–GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function

PubMed Central

Tian, Ji; Pei, Haixia; Ma, Nan

2014-01-01

Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3’ terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV–GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV–GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV–GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75–80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants. PMID:24218330

Efficient and Accurate Algorithm for Cleaved Fragments Prediction (CFPA) in Protein Sequences Dataset Based on Consensus and Its Variants: A Novel Degradomics Prediction Application.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Hajj, Hazem; Kobeissy, Firas H

2017-01-01

Degradomics is a novel discipline that involves determination of the proteases/substrate fragmentation profile, called the substrate degradome, and has been recently applied in different disciplines. A major application of degradomics is its utility in the field of biomarkers where the breakdown products (BDPs) of different protease have been investigated. Among the major proteases assessed, calpain and caspase proteases have been associated with the execution phases of the pro-apoptotic and pro-necrotic cell death, generating caspase/calpain-specific cleaved fragments. The distinction between calpain and caspase protein fragments has been applied to distinguish injury mechanisms. Advanced proteomics technology has been used to identify these BDPs experimentally. However, it has been a challenge to identify these BDPs with high precision and efficiency, especially if we are targeting a number of proteins at one time. In this chapter, we present a novel bioinfromatic detection method that identifies BDPs accurately and efficiently with validation against experimental data. This method aims at predicting the consensus sequence occurrences and their variants in a large set of experimentally detected protein sequences based on state-of-the-art sequence matching and alignment algorithms. After detection, the method generates all the potential cleaved fragments by a specific protease. This space and time-efficient algorithm is flexible to handle the different orientations that the consensus sequence and the protein sequence can take before cleaving. It is O(mn) in space complexity and O(Nmn) in time complexity, with N number of protein sequences, m length of the consensus sequence, and n length of each protein sequence. Ultimately, this knowledge will subsequently feed into the development of a novel tool for researchers to detect diverse types of selected BDPs as putative disease markers, contributing to the diagnosis and treatment of related disorders.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

NASA Astrophysics Data System (ADS)

Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

2014-06-01

Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway.

PubMed

Park, Arnold; Yun, Tatyana; Vigant, Frederic; Pernet, Olivier; Won, Sohui T; Dawes, Brian E; Bartkowski, Wojciech; Freiberg, Alexander N; Lee, Benhur

2016-05-01

The budding of Nipah virus, a deadly member of the Henipavirus genus within the Paramyxoviridae, has been thought to be independent of the host ESCRT pathway, which is critical for the budding of many enveloped viruses. This conclusion was based on the budding properties of the virus matrix protein in the absence of other virus components. Here, we find that the virus C protein, which was previously investigated for its role in antagonism of innate immunity, recruits the ESCRT pathway to promote efficient virus release. Inhibition of ESCRT or depletion of the ESCRT factor Tsg101 abrogates the C enhancement of matrix budding and impairs live Nipah virus release. Further, despite the low sequence homology of the C proteins of known henipaviruses, they all enhance the budding of their cognate matrix proteins, suggesting a conserved and previously unknown function for the henipavirus C proteins.

[Effect of solution environments on ceramic membrane microfiltration of model system of Chinese medicines].

PubMed

Zhang, Lianjun; Lu, Jin; Le, Kang; Fu, Tingming; Guo, Liwei

2010-07-01

To investigate the effect of differents solution environments on the ceramic membrane microfiltration of model system of Chinese medicines. Taking binary system of soybean protein-berberine as the research object, flux, transmittance of berberine and traping rate of protein as indexes, different solution environment on membrane process were investigated. When the concentration of soybean protein was under 1 g x L(-1), the membrane flux was minimum with the traping of berberine decreased slightly as the concentration increased. When pH was 4, the flux was maximum with the traping rate of protein was 99%, and the transmittance of berberine reached above 60%. The efficiency of membrane separation can be improved by optimizing the solution environment of water-extraction of chinese medicines. The efficiency of membrane separation is the best when adjust the pH to the isoelectric point of proteins for the proteins as the main pollutant in aqueous solution.

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

The ability of in vitro antioxidant assays to predict the efficiency of a cod protein hydrolysate and brown seaweed extract to prevent oxidation in marine food model systems.

PubMed

Jónsdóttir, Rósa; Geirsdóttir, Margrét; Hamaguchi, Patricia Y; Jamnik, Polona; Kristinsson, Hordur G; Undeland, Ingrid

2016-04-01

The ability of different in vitro antioxidant assays to predict the efficiency of cod protein hydrolysate (CPH) and Fucus vesiculosus ethyl acetate extract (EA) towards lipid oxidation in haemoglobin-fortified washed cod mince and iron-containing cod liver oil emulsion was evaluated. The progression of oxidation was followed by sensory analysis, lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) in both systems, as well as loss of redness and protein carbonyls in the cod system. The in vitro tests revealed high reducing capacity, high DPPH radical scavenging properties and a high oxygen radical absorbance capacity (ORAC) value of the EA which also inhibited lipid and protein oxidation in the cod model system. The CPH had a high metal chelating capacity and was efficient against oxidation in the cod liver oil emulsion. The results indicate that the F. vesiculosus extract has a potential as an excellent natural antioxidant against lipid oxidation in fish muscle foods while protein hydrolysates are more promising for fish oil emulsions. The usefulness of in vitro assays to predict the antioxidative properties of new natural ingredients in foods thus depends on the knowledge about the food systems, particularly the main pro-oxidants present. © 2015 Society of Chemical Industry.

PSOVina: The hybrid particle swarm optimization algorithm for protein-ligand docking.

PubMed

Ng, Marcus C K; Fong, Simon; Siu, Shirley W I

2015-06-01

Protein-ligand docking is an essential step in modern drug discovery process. The challenge here is to accurately predict and efficiently optimize the position and orientation of ligands in the binding pocket of a target protein. In this paper, we present a new method called PSOVina which combined the particle swarm optimization (PSO) algorithm with the efficient Broyden-Fletcher-Goldfarb-Shannon (BFGS) local search method adopted in AutoDock Vina to tackle the conformational search problem in docking. Using a diverse data set of 201 protein-ligand complexes from the PDBbind database and a full set of ligands and decoys for four representative targets from the directory of useful decoys (DUD) virtual screening data set, we assessed the docking performance of PSOVina in comparison to the original Vina program. Our results showed that PSOVina achieves a remarkable execution time reduction of 51-60% without compromising the prediction accuracies in the docking and virtual screening experiments. This improvement in time efficiency makes PSOVina a better choice of a docking tool in large-scale protein-ligand docking applications. Our work lays the foundation for the future development of swarm-based algorithms in molecular docking programs. PSOVina is freely available to non-commercial users at http://cbbio.cis.umac.mo .

Aggregation Kinetics of Hematite Particles in the Presence of Outer Membrane Cytochrome OmcA of Shewanella oneidenesis MR-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Anxu; Liu, Feng; Shi, Liang

2016-09-20

The aggregation behavior of 9, 36, and 112 nm hematite particles was studied in the presence of OmcA, a bacterial extracellular protein, in aqueous dispersions at pH 5.7 through time-resolved dynamic light scattering, electrophoretic mobility, and circular dichroism spectra, respectively. At low salt concentration, the attachment efficiencies of hematite particles in all sizes first increased, then decreased, and finally remained stable with the increase of OmcA concentration, indicating the dominant interparticle interaction changed along with the increase in the protein-to-particle ratio. Nevertheless, at high salt concentration, the attachment efficiencies of all hematite samples gradually decreased with increasing OmcA concentration, whichmore » can be attributed to increasing steric force. Additionally, the aggregation behavior of OmcA-hematite conjugates was more correlated to total particle-surface area than primary particle size. It was further established that OmcA could stabilize hematite nanoparticles more efficiently than bovine serum albumin (BSA), a model plasma protein, due to the higher affinity of OmcA to hematite surface. This study highlighted the effects of particle properties, solution conditions, and protein properties on the complicated aggregation behavior of protein-nanoparticle conjugates in aqueous environments.« less

Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis

PubMed Central

Tyagi, Anil K.

2017-01-01

Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological roles are less understood. Mycobacterium tuberculosis possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing M. tuberculosis bfr mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell. PMID:28060867

3Drefine: an interactive web server for efficient protein structure refinement

PubMed Central

Bhattacharya, Debswapna; Nowotny, Jackson; Cao, Renzhi; Cheng, Jianlin

2016-01-01

3Drefine is an interactive web server for consistent and computationally efficient protein structure refinement with the capability to perform web-based statistical and visual analysis. The 3Drefine refinement protocol utilizes iterative optimization of hydrogen bonding network combined with atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields for efficient protein structure refinement. The method has been extensively evaluated on blind CASP experiments as well as on large-scale and diverse benchmark datasets and exhibits consistent improvement over the initial structure in both global and local structural quality measures. The 3Drefine web server allows for convenient protein structure refinement through a text or file input submission, email notification, provided example submission and is freely available without any registration requirement. The server also provides comprehensive analysis of submissions through various energy and statistical feedback and interactive visualization of multiple refined models through the JSmol applet that is equipped with numerous protein model analysis tools. The web server has been extensively tested and used by many users. As a result, the 3Drefine web server conveniently provides a useful tool easily accessible to the community. The 3Drefine web server has been made publicly available at the URL: http://sysbio.rnet.missouri.edu/3Drefine/. PMID:27131371

Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.

PubMed

Didenko, Tatiana; Proudfoot, Andrew; Dutta, Samit Kumar; Serrano, Pedro; Wüthrich, Kurt

2015-08-24

High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non-uniform sampling (NUS) of 3D heteronuclear-resolved [(1)H,(1)H]-NOESY data yielded two- to three-fold savings of instrument time for structure determinations of soluble proteins. With the 152-residue protein NP_372339.1 from Staphylococcus aureus and the 71-residue protein NP_346341.1 from Streptococcus pneumonia we show that high-quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BWM*: A Novel, Provable, Ensemble-based Dynamic Programming Algorithm for Sparse Approximations of Computational Protein Design.

PubMed

Jou, Jonathan D; Jain, Swati; Georgiev, Ivelin S; Donald, Bruce R

2016-06-01

Sparse energy functions that ignore long range interactions between residue pairs are frequently used by protein design algorithms to reduce computational cost. Current dynamic programming algorithms that fully exploit the optimal substructure produced by these energy functions only compute the GMEC. This disproportionately favors the sequence of a single, static conformation and overlooks better binding sequences with multiple low-energy conformations. Provable, ensemble-based algorithms such as A* avoid this problem, but A* cannot guarantee better performance than exhaustive enumeration. We propose a novel, provable, dynamic programming algorithm called Branch-Width Minimization* (BWM*) to enumerate a gap-free ensemble of conformations in order of increasing energy. Given a branch-decomposition of branch-width w for an n-residue protein design with at most q discrete side-chain conformations per residue, BWM* returns the sparse GMEC in O([Formula: see text]) time and enumerates each additional conformation in merely O([Formula: see text]) time. We define a new measure, Total Effective Search Space (TESS), which can be computed efficiently a priori before BWM* or A* is run. We ran BWM* on 67 protein design problems and found that TESS discriminated between BWM*-efficient and A*-efficient cases with 100% accuracy. As predicted by TESS and validated experimentally, BWM* outperforms A* in 73% of the cases and computes the full ensemble or a close approximation faster than A*, enumerating each additional conformation in milliseconds. Unlike A*, the performance of BWM* can be predicted in polynomial time before running the algorithm, which gives protein designers the power to choose the most efficient algorithm for their particular design problem.

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.

PubMed

Fan, Chenguang; Xiong, Hai; Reynolds, Noah M; Söll, Dieter

2015-12-15

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA(Pyl) to create tRNA(Pyl-opt) with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N(ϵ)-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA(Pyl-opt) facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA(Pyl-opt) with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA(Pyl-opt) had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA(Pyl-opt) should be an excellent replacement of wild-type tRNA(Pyl) for future ncAA incorporation by PylRS enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Rapid and efficient hydrophilicity tuning of p53/mdm2 antagonists*

PubMed Central

Srivastava, Stuti; Beck, Barbara; Wang, Wei; Czarna, Anna; Holak, Tad A.; Dömling, Alexander

2009-01-01

The protein-protein interaction of p53 and mdm2 is an important anticancer target. The interface, however, is very hydrophobic and naturally results in very hydrophobic antagonists. We used the Orru three component reaction (O-3CR) along with a rapid and efficient, recently discovered amidation reaction to dramatically improve the water solubility of our recently discovered low molecular weight p53/mdm2 antagonists. Arrays of amides were synthesized with improved hydrophilicity and retainment and/or improvement of p53/mdm2 inhibitory activity. PMID:19548636

RELATIVE ACTIN NUCLEATION PROMOTION EFFICIENCY BY WASP AND WAVE PROTEINS IN ENDOTHELIAL CELLS

PubMed Central

Kang, Hyeran; Wang, Jingjing; Longley, Sarah J.; Tang, Jay X.; Shaw, Sunil K.

2010-01-01

The mammalian genome encodes multiple WASP1 (Wiskott-Aldrich Syndrome Protein)/WAVE (WASP-family Verprolin homologous) proteins. Members of this family interact with the Arp (actin related protein) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors. PMID:20816932

Feeding strategy, nitrogen cycling, and profitability of dairy farms.

PubMed

Rotz, C A; Satter, L D; Mertens, D R; Muck, R E

1999-12-01

On a typical dairy farm today, large amounts of N are imported as feed supplements and fertilizer. If this N is not recycled through crop growth, it can lead to large losses to the atmosphere and ground water. More efficient use of protein feed supplements can potentially reduce the import of N in feeds, excretion of N in manure, and losses to the environment. A simulation study with a dairy farm model (DAFOSYM) illustrated that more efficient feeding and use of protein supplements increased farm profit and reduced N loss from the farm. Compared to soybean meal as the sole protein supplement, use of soybean meal along with a less rumen degradable protein feed reduced volatile N loss by 13 to 34 kg/ha of cropland with a small reduction in N leaching loss (about 1 kg/ha). Using the more expensive but less degradable protein supplement along with soybean meal improved net return by $46 to $69/cow per year, dependent on other management strategies of the farm. Environmental and economic benefits from more efficient supplementation of protein were generally greater with more animals per unit of land, higher milk production, more sandy soils, or a daily manure hauling strategy. Relatively less benefit was obtained when either alfalfa or corn silage was the sole forage on the farm or when relatively high amounts of forage were used in animal rations.

Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage.

PubMed

Tornetta, Mark; Baker, Scott; Whitaker, Brian; Lu, Jin; Chen, Qiang; Pisors, Eileen; Shi, Lei; Luo, Jinquan; Sweet, Raymond; Tsui, Ping

2010-08-31

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins. 2010 Elsevier B.V. All rights reserved.

Demonstrating an Order-of-Magnitude Sampling Enhancement in Molecular Dynamics Simulations of Complex Protein Systems.

PubMed

Pan, Albert C; Weinreich, Thomas M; Piana, Stefano; Shaw, David E

2016-03-08

Molecular dynamics (MD) simulations can describe protein motions in atomic detail, but transitions between protein conformational states sometimes take place on time scales that are infeasible or very expensive to reach by direct simulation. Enhanced sampling methods, the aim of which is to increase the sampling efficiency of MD simulations, have thus been extensively employed. The effectiveness of such methods when applied to complex biological systems like proteins, however, has been difficult to establish because even enhanced sampling simulations of such systems do not typically reach time scales at which convergence is extensive enough to reliably quantify sampling efficiency. Here, we obtain sufficiently converged simulations of three proteins to evaluate the performance of simulated tempering, a member of a widely used class of enhanced sampling methods that use elevated temperature to accelerate sampling. Simulated tempering simulations with individual lengths of up to 100 μs were compared to (previously published) conventional MD simulations with individual lengths of up to 1 ms. With two proteins, BPTI and ubiquitin, we evaluated the efficiency of sampling of conformational states near the native state, and for the third, the villin headpiece, we examined the rate of folding and unfolding. Our comparisons demonstrate that simulated tempering can consistently achieve a substantial sampling speedup of an order of magnitude or more relative to conventional MD.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

PubMed

Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

2016-11-01

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

Novel value-added uses for sweet potato juice and flour in polyphenol- and protein-enriched functional food ingredients.

PubMed

Grace, Mary H; Truong, An N; Truong, Van-Den; Raskin, Ilya; Lila, Mary Ann

2015-09-01

Blackcurrant, blueberry, and muscadine grape juices were efficiently sorbed, concentrated, and stabilized into dry granular ingredient matrices which combined anti-inflammatory and antioxidant fruit polyphenols with sweet potato functional constituents (carotenoids, vitamins, polyphenols, fibers). Total phenolics were highest in blackcurrant-orange sweet potato ingredient matrices (34.03 mg/g), and lowest in muscadine grape-yellow sweet potato matrices (10.56 mg/g). Similarly, anthocyanins were most concentrated in blackcurrant-fortified orange and yellow sweet potato matrices (5.40 and 6.54 mg/g, respectively). Alternatively, other protein-rich edible matrices (defatted soy flour, light roasted peanut flour, and rice protein concentrate) efficiently captured polyphenols (6.09-9.46 mg/g) and anthocyanins (0.77-1.27 mg/g) from purple-fleshed sweet potato juice, with comparable efficiency. Antioxidant activity correlated well with total phenolic content. All formulated ingredient matrices stabilized and preserved polyphenols for up to 24 weeks, even when stored at 37°C. Complexation with juice-derived polyphenols did not significantly alter protein or carbohydrate profiles of the matrices. Sensory evaluation of the ingredient matrices suggested potential uses for a wide range of functional food products.

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

NASA Astrophysics Data System (ADS)

Di Rocco, Giulia; Bernini, Fabrizio; Borsari, Marco; Martinelli, Ilaria; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Ranieri, Antonio; Caselli, Monica; Sola, Marco; Ponterini, Glauco

2016-09-01

The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.

Replacing dietary soybean meal with canola meal improves production and efficiency of lactating dairy cows

USDA-ARS?s Scientific Manuscript database

Previous research suggested that crude protein (CP) from canola meal (CM) is used more efficiently than CP from solvent soybean meal (SBM) by lactating dairy cows. We tested whether dietary CP content influenced relative effectiveness of equal supplemental CP from either CM or SBM. Fifty lactating H...

PRODUCTION AND ECONOMIC OPTIMIZATION OF DIETARY PROTEIN AND CARBOHYDRATE IN THE CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus

PubMed Central

Heflin, Laura E.; Makowsky, Robert; Taylor, J. Christopher; Williams, Michael B.; Lawrence, Addison L.; Watts, Stephen A.

2016-01-01

Juvenile Lytechinus variegatus (ca. 3.95± 0.54 g) were fed one of 10 formulated diets with different protein (ranging from 11- 43%) and carbohydrate (12 or 18%; brackets determined from previous studies) levels. Urchins (n= 16 per treatment) were fed a daily sub-satiation ration equivalent to 2.0% of average body weight for 10 weeks. Our objective was (1) to create predictive models of growth, production and efficiency outcomes and (2) to generate economic analysis models in relation to these dietary outcomes for juvenile L. variegatus held in culture. At dietary protein levels below ca. 30%, models for most growth and production outcomes predicted increased rates of growth and production among urchins fed diets containing 18% dietary carbohydrate levels as compared to urchins fed diets containing 12% dietary carbohydrate. For most outcomes, growth and production was predicted to increase with increasing level of dietary protein up to ca. 30%, after which, no further increase in growth and production were predicted. Likewise, dry matter production efficiency was predicted to increase with increasing protein level up to ca. 30%, with urchins fed diets with 18% carbohydrate exhibiting greater efficiency than those fed diets with 12% carbohydrate. The energetic cost of dry matter production was optimal at protein levels less than those required for maximal weight gain and gonad production, suggesting an increased energetic cost (decreased energy efficiency) is required to increase gonad production relative to somatic growth. Economic analysis models predict when cost of feed ingredients are low, the lowest cost per gram of wet weight gain will occur at 18% dietary carbohydrate and ca. 25- 30% dietary protein. In contrast, lowest cost per gram of wet weight gain will occur at 12% dietary carbohydrate and ca. 35- 40% dietary protein when feed ingredient costs are high or average. For both 18 and 12% levels of dietary carbohydrate, cost per gram of wet weight gain is predicted to be maximized at low dietary protein levels, regardless of feed ingredient costs. These models will compare dietary requirements and growth outcomes in relation to economic costs and provide insight for future commercialization of sea urchin aquaculture. PMID:28082753

PRODUCTION AND ECONOMIC OPTIMIZATION OF DIETARY PROTEIN AND CARBOHYDRATE IN THE CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus.

PubMed

Heflin, Laura E; Makowsky, Robert; Taylor, J Christopher; Williams, Michael B; Lawrence, Addison L; Watts, Stephen A

2016-10-01

Juvenile Lytechinus variegatus (ca. 3.95± 0.54 g) were fed one of 10 formulated diets with different protein (ranging from 11- 43%) and carbohydrate (12 or 18%; brackets determined from previous studies) levels. Urchins (n= 16 per treatment) were fed a daily sub-satiation ration equivalent to 2.0% of average body weight for 10 weeks. Our objective was (1) to create predictive models of growth, production and efficiency outcomes and (2) to generate economic analysis models in relation to these dietary outcomes for juvenile L. variegatus held in culture. At dietary protein levels below ca. 30%, models for most growth and production outcomes predicted increased rates of growth and production among urchins fed diets containing 18% dietary carbohydrate levels as compared to urchins fed diets containing 12% dietary carbohydrate. For most outcomes, growth and production was predicted to increase with increasing level of dietary protein up to ca. 30%, after which, no further increase in growth and production were predicted. Likewise, dry matter production efficiency was predicted to increase with increasing protein level up to ca. 30%, with urchins fed diets with 18% carbohydrate exhibiting greater efficiency than those fed diets with 12% carbohydrate. The energetic cost of dry matter production was optimal at protein levels less than those required for maximal weight gain and gonad production, suggesting an increased energetic cost (decreased energy efficiency) is required to increase gonad production relative to somatic growth. Economic analysis models predict when cost of feed ingredients are low, the lowest cost per gram of wet weight gain will occur at 18% dietary carbohydrate and ca. 25- 30% dietary protein. In contrast, lowest cost per gram of wet weight gain will occur at 12% dietary carbohydrate and ca. 35- 40% dietary protein when feed ingredient costs are high or average. For both 18 and 12% levels of dietary carbohydrate, cost per gram of wet weight gain is predicted to be maximized at low dietary protein levels, regardless of feed ingredient costs. These models will compare dietary requirements and growth outcomes in relation to economic costs and provide insight for future commercialization of sea urchin aquaculture.

Cartilage ablation studies using mid-IR free electron laser

NASA Astrophysics Data System (ADS)

Youn, Jong-In; Peavy, George M.; Venugopalan, Vasan

2005-04-01

The ablation rate of articular cartilage and fibrocartilage (meniscus), were quantified to examine wavelength and tissue-composition dependence of ablation efficiency for selected mid-infrared wavelengths. The wavelengths tested were 2.9 um (water dominant absorption), 6.1 (protein and water absorption) and 6.45 um (protein dominant absorption) generated by the Free Electron Laser (FEL) at Vanderbilt University. The measurement of tissue mass removal using a microbalance during laser ablation was conducted to determine the ablation rates of cartilage. The technique can be accurate over methods such as profilometer and histology sectioning where tissue surface and the crater morphology may be affected by tissue processing. The ablation efficiency was found to be dependent upon the wavelength. Both articular cartilage and meniscus (fibrocartilage) ablations at 6.1 um were more efficient than those at the other wavelengths evaluated. We observed the lowest ablation efficiency of both types of cartilage with the 6.45 um wavelength, possibly due to the reduction in water absorption at this wavelength in comparison to the other wavelengths that were evaluated.

Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum.

PubMed

Sandra, Koen; Verleysen, Katleen; Labeur, Christine; Vanneste, Lies; D'Hondt, Filip; Thomas, Grégoire; Kas, Koen; Gevaert, Kris; Vandekerckhove, Joël; Sandra, Pat

2007-03-01

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

PubMed Central

Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

2015-01-01

DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick

2005-07-14

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there ismore » as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.« less

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

PubMed

Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

2011-08-01

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

PubMed Central

Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

2014-01-01

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins. PMID:24390926

Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins

NASA Astrophysics Data System (ADS)

Alam, Rabeka; Zylstra, Joshua; Fontaine, Danielle M.; Branchini, Bruce R.; Maye, Mathew M.

2013-05-01

Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated. Electronic supplementary information (ESI) available: Experimental details, Fig. S1 and Table S1-S4. See DOI: 10.1039/c3nr01842c

Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

PubMed Central

Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

2017-01-01

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

Efficiency of food utilization by pregnant and lactating sows. 2. The influence of isocaloric diets with different protein levels on pregnancy and lactation.

PubMed

Burlacu, G; Iliescu, M; Cărămidă, P

1986-09-01

The efficiency of utilization of feed energy as digestible, metabolizable and net energy is similar in pregnant and lactating sows irrespective of the stage of these physiological conditions. This efficiency with the value of about 71% resembles the one found in the growing and fattening pigs, which enables us to use, for this category of animals, the same system offered evaluation and of the energy requirements based on fat nutritive units. The maintenance energy requirement expressed in ME varies from 467 kJ/kg 0.75 in pregnant sows to 512 kJ/kg 0.75 in lactating sows, for the suckling piglets having intermediary value of 498 kJ/kg 0.75. The efficiency of utilization of diets protein for maintenance and for synthesis is also similar for these categories of pigs, varying from 71% in lactating sows to 75-76% in pregnant sows. In suckling piglets we had recorded an efficiency of DCP utilization which varies parabolically with size of the ingesta and therefore with that of weight gain. The DCP maintenance requirement expressed in g N dig./kg 0.75 varies within narrow limits between 0.345 g N in suckling piglets 0.380 g N in pregnant sows; In lactating sows we have detected an intermediary value of 0.355 g N. Based on these experimental data and also using recent experimental results obtained by Schiemann and Beyer (1984) regarding the energy and N content of the foetuses, of the organs of reproductions and of milk, we could calculate the energy and protein requirements. These requirements when compared with the values used in our country show us higher values of energy in pregnant and lactating sows and lower values of protein for the same categories, including the piglets, where we have also found lower values for the energy too.

Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Lewis A.; Habershon, Scott, E-mail: S.Habershon@warwick.ac.uk

Pigment-protein complexes (PPCs) play a central role in facilitating excitation energy transfer (EET) from light-harvesting antenna complexes to reaction centres in photosynthetic systems; understanding molecular organisation in these biological networks is key to developing better artificial light-harvesting systems. In this article, we combine quantum-mechanical simulations and a network-based picture of transport to investigate how chromophore organization and protein environment in PPCs impacts on EET efficiency and robustness. In a prototypical PPC model, the Fenna-Matthews-Olson (FMO) complex, we consider the impact on EET efficiency of both disrupting the chromophore network and changing the influence of (local and global) environmental dephasing. Surprisingly,more » we find a large degree of resilience to changes in both chromophore network and protein environmental dephasing, the extent of which is greater than previously observed; for example, FMO maintains EET when 50% of the constituent chromophores are removed, or when environmental dephasing fluctuations vary over two orders-of-magnitude relative to the in vivo system. We also highlight the fact that the influence of local dephasing can be strongly dependent on the characteristics of the EET network and the initial excitation; for example, initial excitations resulting in rapid coherent decay are generally insensitive to the environment, whereas the incoherent population decay observed following excitation at weakly coupled chromophores demonstrates a more pronounced dependence on dephasing rate as a result of the greater possibility of local exciton trapping. Finally, we show that the FMO electronic Hamiltonian is not particularly optimised for EET; instead, it is just one of many possible chromophore organisations which demonstrate a good level of EET transport efficiency following excitation at different chromophores. Overall, these robustness and efficiency characteristics are attributed to the highly connected nature of the chromophore network and the presence of multiple EET pathways, features which might easily be built into artificial photosynthetic systems.« less

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

PubMed

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-03-02

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores.

PubMed

Hinc, Krzysztof; Isticato, Rachele; Dembek, Marcin; Karczewska, Joanna; Iwanicki, Adam; Peszyńska-Sularz, Grazyna; De Felice, Maurilio; Obuchowski, Michał; Ricca, Ezio

2010-01-18

The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections. We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed. UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

Energy efficiency of digestible protein, fat and carbohydrate utilisation for growth in rainbow trout and Nile tilapia.

PubMed

Schrama, Johan W; Haidar, Mahmoud N; Geurden, Inge; Heinsbroek, Leon T N; Kaushik, Sachi J

2018-04-01

Currently, energy evaluation of fish feeds is performed on a digestible energy basis. In contrast to net energy (NE) evaluation systems, digestible energy evaluation systems do not differentiate between the different types of digested nutrients regarding their potential for growth. The aim was to develop an NE evaluation for fish by estimating the energy efficiency of digestible nutrients (protein, fat and carbohydrates) and to assess whether these efficiencies differed between Nile tilapia and rainbow trout. Two data sets were constructed. The tilapia and rainbow data set contained, respectively, eight and nine experiments in which the digestibility of protein, fat and energy and the complete energy balances for twenty-three and forty-five diets was measured. The digestible protein (dCP), digestible fat (dFat) and digestible carbohydrate intakes (dCarb) were calculated. By multiple regression analysis, retained energy (RE) was related to dCP, dFat and dCarb. In tilapia, all digestible nutrients were linearly related to RE (P<0·001). In trout, RE was quadratically related to dCarb (P<0·01) and linearly to dCP and dFat (P<0·001). The NE formula was NE=11·5×dCP+35·8×dFAT+11·3×dCarb for tilapia and NE=13·5×dCP+33·0×dFAT+34·0×dCarb-3·64×(dCarb)2 for trout (NE in kJ/(kg0·8×d); dCP, dFat and dCarb in g/(kg0·8×d)). In tilapia, the energetic efficiency of dCP, dFat and dCarb was 49, 91 and 66 %, respectively, showing large similarity with pigs. Tilapia and trout had similar energy efficiencies of dCP (49 v. 57 %) and dFat (91 v. 84 %), but differed regarding dCarb.

Enhanced energy conversion efficiency from high strength synthetic organic wastewater by sequential dark fermentative hydrogen production and algal lipid accumulation.

PubMed

Ren, Hong-Yu; Liu, Bing-Feng; Kong, Fanying; Zhao, Lei; Xing, Defeng; Ren, Nan-Qi

2014-04-01

A two-stage process of sequential dark fermentative hydrogen production and microalgal cultivation was applied to enhance the energy conversion efficiency from high strength synthetic organic wastewater. Ethanol fermentation bacterium Ethanoligenens harbinense B49 was used as hydrogen producer, and the energy conversion efficiency and chemical oxygen demand (COD) removal efficiency reached 18.6% and 28.3% in dark fermentation. Acetate was the main soluble product in dark fermentative effluent, which was further utilized by microalga Scenedesmus sp. R-16. The final algal biomass concentration reached 1.98gL(-1), and the algal biomass was rich in lipid (40.9%) and low in protein (23.3%) and carbohydrate (11.9%). Compared with single dark fermentation stage, the energy conversion efficiency and COD removal efficiency of two-stage system remarkably increased 101% and 131%, respectively. This research provides a new approach for efficient energy production and wastewater treatment using a two-stage process combining dark fermentation and algal cultivation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Estimating and testing interactions when explanatory variables are subject to non-classical measurement error.

PubMed

Murad, Havi; Kipnis, Victor; Freedman, Laurence S

2016-10-01

Assessing interactions in linear regression models when covariates have measurement error (ME) is complex.We previously described regression calibration (RC) methods that yield consistent estimators and standard errors for interaction coefficients of normally distributed covariates having classical ME. Here we extend normal based RC (NBRC) and linear RC (LRC) methods to a non-classical ME model, and describe more efficient versions that combine estimates from the main study and internal sub-study. We apply these methods to data from the Observing Protein and Energy Nutrition (OPEN) study. Using simulations we show that (i) for normally distributed covariates efficient NBRC and LRC were nearly unbiased and performed well with sub-study size ≥200; (ii) efficient NBRC had lower MSE than efficient LRC; (iii) the naïve test for a single interaction had type I error probability close to the nominal significance level, whereas efficient NBRC and LRC were slightly anti-conservative but more powerful; (iv) for markedly non-normal covariates, efficient LRC yielded less biased estimators with smaller variance than efficient NBRC. Our simulations suggest that it is preferable to use: (i) efficient NBRC for estimating and testing interaction effects of normally distributed covariates and (ii) efficient LRC for estimating and testing interactions for markedly non-normal covariates. © The Author(s) 2013.

Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins

PubMed Central

Rezelj, Veronica V.; Li, Ping; Chaudhary, Vidyanath; Elliott, Richard M.

2017-01-01

ABSTRACT In recent years, several newly discovered tick-borne viruses causing a wide spectrum of diseases in humans have been ascribed to the Phlebovirus genus of the Bunyaviridae family. The nonstructural protein (NSs) of bunyaviruses is the main virulence factor and interferon (IFN) antagonist. We studied the molecular mechanisms of IFN antagonism employed by the NSs proteins of human apathogenic Uukuniemi virus (UUKV) and those of Heartland virus (HRTV) and severe fever with thrombocytopenia syndrome virus (SFTSV), both of which cause severe disease. Using reporter assays, we found that UUKV NSs weakly inhibited the activation of the beta interferon (IFN-β) promoter and response elements. UUKV NSs weakly antagonized human IFN-β promoter activation through a novel interaction with mitochondrial antiviral-signaling protein (MAVS), confirmed by coimmunoprecipitation and confocal microscopy studies. HRTV NSs efficiently antagonized both IFN-β promoter activation and type I IFN signaling pathways through interactions with TBK1, preventing its phosphorylation. HRTV NSs exhibited diffused cytoplasmic localization. This is in comparison to the inclusion bodies formed by SFTSV NSs. HRTV NSs also efficiently interacted with STAT2 and impaired IFN-β-induced phosphorylation but did not affect STAT1 or its translocation to the nucleus. Our results suggest that a weak interaction between STAT1 and HRTV or SFTSV NSs may explain their inability to block type II IFN signaling efficiently, thus enabling the activation of proinflammatory responses that lead to severe disease. Our findings offer insights into how pathogenicity may be linked to the capacity of NSs proteins to block the innate immune system and illustrate the plethora of viral immune evasion strategies utilized by emerging phleboviruses. IMPORTANCE Since 2011, there has been a large expansion in the number of emerging tick-borne viruses that have been assigned to the Phlebovirus genus. Heartland virus (HRTV) and SFTS virus (SFTSV) were found to cause severe disease in humans, unlike other documented tick-borne phleboviruses such as Uukuniemi virus (UUKV). Phleboviruses encode nonstructural proteins (NSs) that enable them to counteract the human innate antiviral defenses. We assessed how these proteins interacted with the innate immune system. We found that UUKV NSs engaged with innate immune factors only weakly, at one early step. However, the viruses that cause more severe disease efficiently disabled the antiviral response by targeting multiple components at several stages across the innate immune induction and signaling pathways. Our results suggest a correlation between the efficiency of the virus protein/host interaction and severity of disease. PMID:28680969

Role of messenger RNA-ribosome complex in complementary DNA display.

PubMed

Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

2013-07-15

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. Copyright © 2013 Elsevier Inc. All rights reserved.

Grain sorghum is a viable feedstock for ethanol production.

PubMed

Wang, D; Bean, S; McLaren, J; Seib, P; Madl, R; Tuinstra, M; Shi, Y; Lenz, M; Wu, X; Zhao, R

2008-05-01

Sorghum is a major cereal crop in the USA. However, sorghum has been underutilized as a renewable feedstock for bioenergy. The goal of this research was to improve the bioconversion efficiency for biofuels and biobased products from processed sorghum. The main focus was to understand the relationship among "genetics-structure-function-conversion" and the key factors impacting ethanol production, as well as to develop an energy life cycle analysis model (ELCAM) to quantify and prioritize the saving potential from factors identified in this research. Genetic lines with extremely high and low ethanol fermentation efficiency and some specific attributes that may be manipulated to improve the bioconversion rate of sorghum were identified. In general, ethanol yield increased as starch content increased. However, no linear relationship between starch content and fermentation efficiency was found. Key factors affecting the ethanol fermentation efficiency of sorghum include protein digestibility, level of extractable proteins, protein and starch interaction, mash viscosity, amount of phenolic compounds, ratio of amylose to amylopectin, and formation of amylose-lipid complexes in the mash. A platform ELCAM with a base case showed a positive net energy value (NEV) = 25,500 Btu/gal EtOH. ELCAM cases were used to identify factors that most impact sorghum use. For example, a yield increase of 40 bu/ac resulted in NEV increasing from 7 million to 12 million Btu/ac. An 8% increase in starch provided an incremental 1.2 million Btu/ac.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation.

PubMed

Massahi, Aslan; Çalık, Pınar

2016-11-07

For extracellular recombinant protein production, the efficiency of five endogenous secretion signal peptides (SPs) of Pichia pastoris, SP13 (MLSTILNIFILLLFIQASLQ), SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), SP34 (MRPVLSLLLLLASSVLA), selected based on their D-score which quantifies the signal peptide-ness of a given sequence segment, was investigated using recombinant human growth hormone (rhGH) as the model protein. The expression was conducted under glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). The highest secretion efficiency among endogenous SPs was obtained by SP23 followed by SP24, SP34, SP13 and SP26, respectively. The fermentation characteristics of rhGH production by the use of SP23, the most favorable endogenous SP of P.pastoris, and Saccharomyces cerevisiae α-mating factor prepro sequence (α-MF) were compared. With respect to the SP23 which is 73 amino acids shorter in length compared to α-MF, in high cell density cultures, where carbon and energy source are limited, the substitution of SP23 for α-MF seems promising. α-MF higher secretion efficiency was verified by major physicochemical properties including hydropathy index, isoelectric point, and aliphatic index. Regarding the examined endogenous SPs, there was no clear correlation between secretion efficiency and major physicochemical properties when each of these properties was considered independently. To find a correlation, factors such as protein N-terminus effect, length of the SP, secondary structure of the SP, and interactions of the selected properties should also be investigated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial efficiency and exercise economy following heat stress: a potential role of uncoupling protein 3.

PubMed

Salgado, Roy M; Sheard, Ailish C; Vaughan, Roger A; Parker, Daryl L; Schneider, Suzanne M; Kenefick, Robert W; McCormick, James J; Gannon, Nicholas P; Van Dusseldorp, Trisha A; Kravitz, Len R; Mermier, Christine M

2017-02-01

Heat stress has been reported to reduce uncoupling proteins (UCP) expression, which in turn should improve mitochondrial efficiency. Such an improvement in efficiency may translate to the systemic level as greater exercise economy. However, neither the heat-induced improvement in mitochondrial efficiency (due to decrease in UCP), nor its potential to improve economy has been studied. Determine: (i) if heat stress in vitro lowers UCP3 thereby improving mitochondrial efficiency in C2C12 myocytes; (ii) whether heat acclimation (HA) in vivo improves exercise economy in trained individuals; and (iii) the potential improved economy during exercise at altitude. In vitro, myocytes were heat stressed for 24 h (40°C), followed by measurements of UCP3, mitochondrial uncoupling, and efficiency. In vivo, eight trained males completed: (i) pre-HA testing; (ii) 10 days of HA (40°C, 20% RH); and (iii) post-HA testing. Pre- and posttesting consisted of maximal exercise test and submaximal exercise at two intensities to assess exercise economy at 1600 m (Albuquerque, NM) and 4350 m. Heat-stressed myocytes displayed significantly reduced UCP3 mRNA expression and, mitochondrial uncoupling (77.1 ± 1.2%, P < 0.0001) and improved mitochondrial efficiency (62.9 ± 4.1%, P < 0.0001) compared to control. In humans, at both 1600 m and 4350 m, following HA, submaximal exercise economy did not change at low and moderate exercise intensities. Our findings indicate that while heat-induced reduction in UCP3 improves mitochondrial efficiency in vitro, this is not translated to in vivo improvement of exercise economy at 1600 m or 4350 m. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Factors affecting energy and nitrogen efficiency of dairy cows: a meta-analysis.

PubMed

Phuong, H N; Friggens, N C; de Boer, I J M; Schmidely, P

2013-01-01

A meta-analysis was performed to explore the correlation between energy and nitrogen efficiency of dairy cows, and to study nutritional and animal factors that influence these efficiencies, as well as their relationship. Treatment mean values were extracted from 68 peer-reviewed studies, including 306 feeding trials. The main criterion for inclusion of a study in the meta-analysis was that it reported, or permitted calculation of, energy efficiency (Eeff; energy in milk/digestible energy intake) and nitrogen efficiency (Neff; nitrogen in milk/digestible nitrogen intake) at the digestible level (digestible energy or digestible protein). The effect of nutritional and animal variables, including neutral detergent fiber, acid detergent fiber (ADF), digestible energy, digestible protein, proportion of concentrate (PCO), dry matter intake, milk yield, days in milk, and body weight, on Eeff, Neff, and the Neff:Eeff ratio was analyzed using mixed models. The interstudy correlation between Eeff and Neff was 0.62, whereas the intrastudy correlation was 0.30. The higher interstudy correlation was partly due to milk yield and dry matter intake being present in both Eeff and Neff. We, therefore, also explored the Neff:Eeff ratio. Energy efficiency was negatively associated with ADF and PCO, whereas Neff was negatively associated with ADF and digestible energy. The Neff:Eeff ratio was affected by ADF and PCO only. In conclusion, the results indicate a possibility to maximize feed efficiency in terms of both energy and nitrogen at the same time. In other words, an improvement in Eeff would also mean an improvement in Neff. The current study also shows that these types of transverse data are not sufficient to study the effect of animal factors, such as days in milk, on feed efficiency. Longitudinal measurements per animal would probably be more appropriate. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

PubMed

Batista, Fernanda A H; Almeida, Glessler S; Seraphim, Thiago V; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

2015-01-01

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. © 2014 FEBS.

On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors.

PubMed

Oliveira-Souza, Wellington P; Bronze, Fellipe; Broos, Jaap; Marcondes, Marcelo F M; Oliveira, Vitor

2017-10-21

Biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery. 5-hydroxy-tryptophan (5OH-Trp) can be bio-incorporated using E. coli as expression host however; we have experienced very low incorporation yields - amount of protein containing regular Trp/amount of protein containing the Trp analog - during expressions of 5OH-Trp labeled proteins. Furthermore, this low incorporation yield were verified especially when the widely-used vectors based on the T7 RNA polymerase were used. Testing different 5OH-Trp incorporation protocols we verified that in these T7-based systems, the production of the T7 RNA polymerase is driven by the same elements - lac promoter/IPTG - as the target protein. Consequently, the bio-incorporation of the 5OH-Trp residues also occurs in this crucial enzyme, but, the produced T7 RNA polymerase labeled with 5OH-Trp is inactive or much less active. In the present work, we describe an efficient method to overcome this mentioned problem and bio-incorporate 5OH-Trp in proteins expressed in E. coli., using vectors based on the T7 RNA polymerase-T7 promoter. The two-step induction protocol here described showed incorporation efficiencies of 5OH-Trp higher than 90%. Copyright © 2017 Elsevier Inc. All rights reserved.

Roles of diet protein and temperature in the growth and nutritional energetics of juvenile slider turtles, Trachemys scripta

USGS Publications Warehouse

Avery, Harold W.; Spotila, James R.; Congdon, Justin D.; Fischer, Robert U.; Standora, Edward A.; Avery, Susan B.

1993-01-01

We determined the effects of dietary protein and Ta on growth rates, food consumption rates, digestion rates, and digestive efficiencies of juvenile slider turtles (Trachemys scripta). Results from this study provide a clearer understanding of how these environmental factors interact in influencing body sizes and growth rates of individuals in wild slider turtle populations. Changes in plastron length, carapace length, and body mass were significantly greater for T. scripta eating 25% and 40% crude protein diets than for those eating 10% crude protein. Those consuming 10% crude protein showed significant decreases in body mass and plastron length over a 13-wk period. Individuals at Ta's of 15°, 22°, 28°, or 34° C had food ingestion rates (kJ wk⁻¹) that increased markedly with an increase in Ta. Increasing dietary crude protein concentration increased turtle ingestion rates and influenced the positive effect of Ta. Increasing dietary crude protein concentration alone did not significantly affect turtle consumption rates but did significantly influence the positive effect of Ta. Digestive efficiencies were very high (because of the pelleted diet). Those turtles that ate at 15° C had a digestive efciency of 99.5%, as compared with 98.3% at 22° C, 94.8% at 28° C, and 95.8% at 34° C. Dietary protein concentration did not influence the digestive efficiencies of T. scripta. These data suggest that dietary protein is an important nutritional component to the growth of juvenile slider turtles and that elevated thermal conditions, combined with a high dietary protein availability, may explain the very high growth rates of slider turtles in some wild populations.

On the Origin of Protein Superfamilies and Superfolds

NASA Astrophysics Data System (ADS)

Magner, Abram; Szpankowski, Wojciech; Kihara, Daisuke

2015-02-01

Distributions of protein families and folds in genomes are highly skewed, having a small number of prevalent superfamiles/superfolds and a large number of families/folds of a small size. Why are the distributions of protein families and folds skewed? Why are there only a limited number of protein families? Here, we employ an information theoretic approach to investigate the protein sequence-structure relationship that leads to the skewed distributions. We consider that protein sequences and folds constitute an information theoretic channel and computed the most efficient distribution of sequences that code all protein folds. The identified distributions of sequences and folds are found to follow a power law, consistent with those observed for proteins in nature. Importantly, the skewed distributions of sequences and folds are suggested to have different origins: the skewed distribution of sequences is due to evolutionary pressure to achieve efficient coding of necessary folds, whereas that of folds is based on the thermodynamic stability of folds. The current study provides a new information theoretic framework for proteins that could be widely applied for understanding protein sequences, structures, functions, and interactions.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Quantitative measurement of protein digestion in simulated gastric fluid.

PubMed

Herman, Rod A; Korjagin, Valerie A; Schafer, Barry W

2005-04-01

The digestibility of novel proteins in simulated gastric fluid is considered to be an indicator of reduced risk of allergenic potential in food, and estimates of digestibility for transgenic proteins expressed in crops are required for making a human-health risk assessment by regulatory authorities. The estimation of first-order rate constants for digestion under conditions of low substrate concentration was explored for two protein substrates (azocoll and DQ-ovalbumin). Data conformed to first-order kinetics, and half-lives were relatively insensitive to significant variations in both substrate and pepsin concentration when high purity pepsin preparations were used. Estimation of digestion efficiency using densitometric measurements of relative protein concentration based on SDS-PAGE corroborated digestion estimates based on measurements of dye or fluorescence release from the labeled substrates. The suitability of first-order rate constants for estimating the efficiency of the pepsin digestion of novel proteins is discussed. Results further support a kinetic approach as appropriate for comparing the digestibility of proteins in simulated gastric fluid.

Theranostic potential of gold nanoparticle-protein agglomerates

NASA Astrophysics Data System (ADS)

Sanpui, Pallab; Paul, Anumita; Chattopadhyay, Arun

2015-11-01

Owing to the ever-increasing applications, glittered with astonishing success of gold nanoparticles (Au NPs) in biomedical research as diagnostic and therapeutic agents, the study of Au NP-protein interaction seems critical for maximizing their theranostic efficiency, and thus demands comprehensive understanding. The mutual interaction of Au NPs and proteins at physiological conditions may result in the aggregation of protein, which can ultimately lead to the formation of Au NP-protein agglomerates. In the present article, we try to appreciate the plausible steps involved in the Au NP-induced aggregation of proteins and also the importance of the proteins' three-dimensional structures in the process. The Au NP-protein agglomerates can potentially be exploited for efficient loading and subsequent release of various therapeutically important molecules, including anticancer drugs, with the unique opportunity of incorporating hydrophilic as well as hydrophobic drugs in the same nanocarrier system. Moreover, the Au NP-protein agglomerates can act as `self-diagnostic' systems, allowing investigation of the conformational state of the associated protein(s) as well as the protein-protein or protein-Au NP interaction within the agglomerates. Furthermore, the potential of these Au NP-protein agglomerates as a novel platform for multifunctional theranostic application along with exciting future-possibilities is highlighted here.

Protein pharmacophore selection using hydration-site analysis

PubMed Central

Hu, Bingjie; Lill, Markus A.

2012-01-01

Virtual screening using pharmacophore models is an efficient method to identify potential lead compounds for target proteins. Pharmacophore models based on protein structures are advantageous because a priori knowledge of active ligands is not required and the models are not biased by the chemical space of previously identified actives. However, in order to capture most potential interactions between all potentially binding ligands and the protein, the size of the pharmacophore model, i.e. number of pharmacophore elements, is typically quite large and therefore reduces the efficiency of pharmacophore based screening. We have developed a new method to select important pharmacophore elements using hydration-site information. The basic premise is that ligand functional groups that replace water molecules in the apo protein contribute strongly to the overall binding affinity of the ligand, due to the additional free energy gained from releasing the water molecule into the bulk solvent. We computed the free energy of water released from the binding site for each hydration site using thermodynamic analysis of molecular dynamics (MD) simulations. Pharmacophores which are co-localized with hydration sites with estimated favorable contributions to the free energy of binding are selected to generate a reduced pharmacophore model. We constructed reduced pharmacophore models for three protein systems and demonstrated good enrichment quality combined with high efficiency. The reduction in pharmacophore model size reduces the required screening time by a factor of 200–500 compared to using all protein pharmacophore elements. We also describe a training process using a small set of known actives to reliably select the optimal set of criteria for pharmacophore selection for each protein system. PMID:22397751

Crickets Are Not a Free Lunch: Protein Capture from Scalable Organic Side-Streams via High-Density Populations of Acheta domesticus

PubMed Central

Lundy, Mark E.; Parrella, Michael P.

2015-01-01

It has been suggested that the ecological impact of crickets as a source of dietary protein is less than conventional forms of livestock due to their comparatively efficient feed conversion and ability to consume organic side-streams. This study measured the biomass output and feed conversion ratios of house crickets (Acheta domesticus) reared on diets that varied in quality, ranging from grain-based to highly cellulosic diets. The measurements were made at a much greater population scale and density than any previously reported in the scientific literature. The biomass accumulation was strongly influenced by the quality of the diet (p<0.001), with the nitrogen (N) content, the ratio of N to acid detergent fiber (ADF) content, and the crude fat (CF) content (y=N/ADF+CF) explaining most of the variability between feed treatments (p = 0.02; R2 = 0.96). In addition, for populations of crickets that were able to survive to a harvestable size, the feed conversion ratios measured were higher (less efficient) than those reported from studies conducted at smaller scales and lower population densities. Compared to the industrial-scale production of chickens, crickets fed a poultry feed diet showed little improvement in protein conversion efficiency, a key metric in determining the ecological footprint of grain-based livestock protein. Crickets fed the solid filtrate from food waste processed at an industrial scale via enzymatic digestion were able to reach a harvestable size and achieve feed and protein efficiencies similar to that of chickens. However, crickets fed minimally-processed, municipal-scale food waste and diets composed largely of straw experienced >99% mortality without reaching a harvestable size. Therefore, the potential for A. domesticus to sustainably supplement the global protein supply, beyond what is currently produced via grain-fed chickens, will depend on capturing regionally scalable organic side-streams of relatively high-quality that are not currently being used for livestock production. PMID:25875026

Crickets are not a free lunch: protein capture from scalable organic side-streams via high-density populations of Acheta domesticus.

PubMed

Lundy, Mark E; Parrella, Michael P

2015-01-01

It has been suggested that the ecological impact of crickets as a source of dietary protein is less than conventional forms of livestock due to their comparatively efficient feed conversion and ability to consume organic side-streams. This study measured the biomass output and feed conversion ratios of house crickets (Acheta domesticus) reared on diets that varied in quality, ranging from grain-based to highly cellulosic diets. The measurements were made at a much greater population scale and density than any previously reported in the scientific literature. The biomass accumulation was strongly influenced by the quality of the diet (p<0.001), with the nitrogen (N) content, the ratio of N to acid detergent fiber (ADF) content, and the crude fat (CF) content (y=N/ADF+CF) explaining most of the variability between feed treatments (p = 0.02; R2 = 0.96). In addition, for populations of crickets that were able to survive to a harvestable size, the feed conversion ratios measured were higher (less efficient) than those reported from studies conducted at smaller scales and lower population densities. Compared to the industrial-scale production of chickens, crickets fed a poultry feed diet showed little improvement in protein conversion efficiency, a key metric in determining the ecological footprint of grain-based livestock protein. Crickets fed the solid filtrate from food waste processed at an industrial scale via enzymatic digestion were able to reach a harvestable size and achieve feed and protein efficiencies similar to that of chickens. However, crickets fed minimally-processed, municipal-scale food waste and diets composed largely of straw experienced >99% mortality without reaching a harvestable size. Therefore, the potential for A. domesticus to sustainably supplement the global protein supply, beyond what is currently produced via grain-fed chickens, will depend on capturing regionally scalable organic side-streams of relatively high-quality that are not currently being used for livestock production.

Protein arginine methylation of Npl3 promotes splicing of the SUS1 intron harboring non-consensus 5' splice site and branch site.

PubMed

Muddukrishna, Bhavana; Jackson, Christopher A; Yu, Michael C

2017-06-01

Protein arginine methylation occurs on spliceosomal components and spliceosome-associated proteins, but how this modification contributes to their function in pre-mRNA splicing remains sparse. Here we provide evidence that protein arginine methylation of the yeast SR-/hnRNP-like protein Npl3 plays a role in facilitating efficient splicing of the SUS1 intron that harbors a non-consensus 5' splice site and branch site. In yeast cells lacking the major protein arginine methyltransferase HMT1, we observed a change in the co-transcriptional recruitment of the U1 snRNP subunit Snp1 and Npl3 to pre-mRNAs harboring both consensus (ECM33 and ASC1) and non-consensus (SUS1) 5' splice site and branch site. Using an Npl3 mutant that phenocopies wild-type Npl3 when expressed in Δhmt1 cells, we showed that the arginine methylation of Npl3 is responsible for this. Examination of pre-mRNA splicing efficiency in these mutants reveals the requirement of Npl3 methylation for the efficient splicing of SUS1 intron 1, but not of ECM33 or ASC1. Changing the 5' splice site and branch site in SUS1 intron 1 to the consensus form restored splicing efficiency in an Hmt1-independent manner. Results from biochemical studies show that methylation of Npl3 promotes its optimal association with the U1 snRNP through its association with the U1 snRNP subunit Mud1. Based on these data, we propose a model in which Hmt1, via arginine methylation of Npl3, facilitates U1 snRNP engagement with the pre-mRNA to promote usage of non-consensus splice sites by the splicing machinery. Published by Elsevier B.V.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

PubMed

Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

PubMed Central

Lin, Yii-Lih; Huang, Yen-Jun; Teerapanich, Pattamon; Leïchlé, Thierry

2016-01-01

Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1. PMID:27375819

Growing Chlorella sp. on meat processing wastewater for nutrient removal and biomass production.

PubMed

Lu, Qian; Zhou, Wenguang; Min, Min; Ma, Xiaochen; Chandra, Ceria; Doan, Yen T T; Ma, Yiwei; Zheng, Hongli; Cheng, Sibo; Griffith, Richard; Chen, Paul; Chen, Chi; Urriola, Pedro E; Shurson, Gerald C; Gislerød, Hans R; Ruan, Roger

2015-12-01

In this work, Chlorella sp. (UM6151) was selected to treat meat processing wastewater for nutrient removal and biomass production. To balance the nutrient profile and improve biomass yield at low cost, an innovative algae cultivation model based on wastewater mixing was developed. The result showed that biomass yield (0.675-1.538 g/L) of algae grown on mixed wastewater was much higher than that on individual wastewater and artificial medium. Wastewater mixing eased the bottleneck for algae growth and contributed to the improved biomass yield. Furthermore, in mixed wastewater with sufficient nitrogen, ammonia nitrogen removal efficiencies (68.75-90.38%) and total nitrogen removal efficiencies (30.06-50.94%) were improved. Wastewater mixing also promoted the synthesis of protein in algal cells. Protein content of algae growing on mixed wastewater reached 60.87-68.65%, which is much higher than that of traditional protein source. Algae cultivation model based on wastewater mixing is an efficient and economical way to improve biomass yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficient Exploitation of Separation Space in Two-Dimensional Liquid Chromatography System for Comprehensive and Efficient Proteomic Analyses.

PubMed

Lee, Hangyeore; Mun, Dong-Gi; So, Jeong Eun; Bae, Jingi; Kim, Hokeun; Masselon, Christophe; Lee, Sang-Won

2016-12-06

Proteomics aims to achieve complete profiling of the protein content and protein modifications in cells, tissues, and biofluids and to quantitatively determine changes in their abundances. This information serves to elucidate cellular processes and signaling pathways and to identify candidate protein biomarkers and/or therapeutic targets. Analyses must therefore be both comprehensive and efficient. Here, we present a novel online two-dimensional reverse-phase/reverse-phase liquid chromatography separation platform, which is based on a newly developed online noncontiguous fractionating and concatenating device (NCFC fractionator). In bottom-up proteomics analyses of a complex proteome, this system provided significantly improved exploitation of the separation space of the two RPs, considerably increasing the numbers of peptides identified compared to a contiguous 2D-RP/RPLC method. The fully automated online 2D-NCFC-RP/RPLC system bypassed a number of labor-intensive manual processes required with the previously described offline 2D-NCFC RP/RPLC method, and thus, it offers minimal sample loss in a context of highly reproducible 2D-RP/RPLC experiments.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2015-11-24

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2013-04-09

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Quantitating protein synthesis, degradation, and endogenous antigen processing.

PubMed

Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

2003-03-01

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Effect of dietary taurine supplementation on growth, feed efficiency, and nutrient composition of juvenile sablefish (Anoplopoma fimbria)

USDA-ARS?s Scientific Manuscript database

Juvenile sablefish were fed a low taurine, basal feed with seven graded levels of supplemental taurine to determine taurine requirements for growth and feed efficiency. The basal feed was plant based, formulated primarily with soy and corn proteins with a minimal (9%) amount of fishmeal. The unsuppl...

Genetic improvement of feed conversion ratio via indirect selection against lipid deposition in farmed rainbow trout (Oncorhynchus mykiss Walbaum).

PubMed

Kause, Antti; Kiessling, Anders; Martin, Samuel A M; Houlihan, Dominic; Ruohonen, Kari

2016-11-01

In farmed fish, selective breeding for feed conversion ratio (FCR) may be possible via indirectly selecting for easily-measured indicator traits correlated with FCR. We tested the hypothesis that rainbow trout with low lipid% have genetically better FCR, and that lipid% may be genetically related to retention efficiency of macronutrients, making lipid% a useful indicator trait. A quantitative genetic analysis was used to quantify the benefit of replacing feed intake in a selection index with one of three lipid traits: body lipid%, muscle lipid% or viscera% weight of total body weight (reflecting visceral lipid). The index theory calculations showed that simultaneous selection for weight gain and against feed intake (direct selection to improve FCR) increased the expected genetic response in FCR by 1·50-fold compared with the sole selection for growth. Replacing feed intake in the selection index with body lipid%, muscle lipid% or viscera% increased genetic response in FCR by 1·29-, 1·49- and 1·02-fold, respectively, compared with the sole selection for growth. Consequently, indirect selection for weight gain and against muscle lipid% was almost as effective as direct selection for FCR. Fish with genetically low body and muscle lipid% were more efficient in turning ingested protein into protein weight gain. Both physiological and genetic mechanisms promote the hypothesis that low-lipid% fish are more efficient. These results highlight that in breeding programmes of rainbow trout, control of lipid deposition improves not only FCR but also protein-retention efficiency. This improves resource efficiency of aquaculture and reduces nutrient load to the environment.

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

NASA Astrophysics Data System (ADS)

Bahreini, Elham; Aghaiypour, Khosrow; Abbasalipourkabir, Roghayeh; Mokarram, Ali Rezaei; Goodarzi, Mohammad Taghi; Saidijam, Massoud

2014-07-01

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene ( ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.

Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR.

PubMed

Campos, Maria Jorge; Quesada, Alberto

2017-01-01

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3'-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5'-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5'-end degenerate primers.

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

PubMed Central

Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

2004-01-01

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

Supramolecularly Engineered Circular Bivalent Aptamer for Enhanced Functional Protein Delivery.

PubMed

Jiang, Ying; Pan, Xiaoshu; Chang, Jin; Niu, Weijia; Hou, Weijia; Kuai, Hailan; Zhao, Zilong; Liu, Ji; Wang, Ming; Tan, Weihong

2018-06-06

Circular bivalent aptamers (cb-apt) comprise an emerging class of chemically engineered aptamers with substantially improved stability and molecular recognition ability. Its therapeutic application, however, is challenged by the lack of functional modules to control the interactions of cb-apt with therapeutics. We present the design of a β-cyclodextrin-modified cb-apt (cb-apt-βCD) and its supramolecular interaction with molecular therapeutics via host-guest chemistry for targeted intracellular delivery. The supramolecular ensemble exhibits high serum stability and enhanced intracellular delivery efficiency compared to a monomeric aptamer. The cb-apt-βCD ensemble delivers green fluorescent protein into targeted cells with efficiency as high as 80%, or cytotoxic saporin to efficiently inhibit tumor cell growth. The strategy of conjugating βCD to cb-apt, and subsequently modulating the supramolecular chemistry of cb-apt-βCD, provides a general platform to expand and diversify the function of aptamers, enabling new biological and therapeutic applications.

Group Additivity in Ligand Binding Affinity: An Alternative Approach to Ligand Efficiency.

PubMed

Reynolds, Charles H; Reynolds, Ryan C

2017-12-26

Group additivity is a concept that has been successfully applied to a variety of thermochemical and kinetic properties. This includes drug discovery, where functional group additivity is often assumed in ligand binding. Ligand efficiency can be recast as a special case of group additivity where ΔG/HA is the group equivalent (HA is the number of non-hydrogen atoms in a ligand). Analysis of a large data set of protein-ligand binding affinities (K i ) for diverse targets shows that in general ligand binding is distinctly nonlinear. It is possible to create a group equivalent scheme for ligand binding, but only in the context of closely related proteins, at least with regard to size. This finding has broad implications for drug design from both experimental and computational points of view. It also offers a path forward for a more general scheme to assess the efficiency of ligand binding.

Biodegradable protein-based rockets for drug transportation and light-triggered release.

PubMed

Wu, Zhiguang; Lin, Xiankun; Zou, Xian; Sun, Jianmin; He, Qiang

2015-01-14

We describe a biodegradable, self-propelled bovine serum albumin/poly-l-lysine (PLL/BSA) multilayer rocket as a smart vehicle for efficient anticancer drug encapsulation/delivery to cancer cells and near-infrared light controlled release. The rockets were constructed by a template-assisted layer-by-layer assembly of the PLL/BSA layers, followed by incorporation of a heat-sensitive gelatin hydrogel containing gold nanoparticles, doxorubicin, and catalase. These rockets can rapidly deliver the doxorubicin to the targeted cancer cell with a speed of up to 68 μm/s, through a combination of biocatalytic bubble propulsion and magnetic guidance. The photothermal effect of the gold nanoparticles under NIR irradiation enable the phase transition of the gelatin hydrogel for rapid release of the loaded doxorubicin and efficient killing of the surrounding cancer cells. Such biodegradable and multifunctional protein-based microrockets provide a convenient and efficient platform for the rapid delivery and controlled release of therapeutic drugs.

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

NASA Astrophysics Data System (ADS)

Li, Cuiping; Yu, Huahua; Feng, Jinhua; Chen, Xiaolin; Li, Pengcheng

2009-02-01

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.

Design and engineering of water-soluble light-harvesting protein maquettes

DOE PAGES

Kodali, Goutham; Mancini, Joshua A.; Solomon, Lee A.; ...

2017-01-01

Design of nanometer scale artificial light harvesting and charge separating proteins enables reengineering to overcome the limitations of natural selection for efficient systems that better meet human energetic needs.

Design and engineering of water-soluble light-harvesting protein maquettes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kodali, Goutham; Mancini, Joshua A.; Solomon, Lee A.

Design of nanometer scale artificial light harvesting and charge separating proteins enables reengineering to overcome the limitations of natural selection for efficient systems that better meet human energetic needs.

Engineering cholesterol-based fibers for antibody immobilization and cell capture

NASA Astrophysics Data System (ADS)

Cohn, Celine

In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing abilities of the CSS fibers were compared to that of hydrophobic polycaprolactone (PCL) fibers and hydrophilic plasma-treated PCL fibers. Electrospun CSS fibers were found to immobilize equivalent amounts of protein as hydrophobically immobilized proteins. However, these proteins captured 6 times more cells, indicative of retained protein function. The second key concept was the design and fabrication of a hybridized lipid fiber. Lipid fibers provide improved protein function but fabrication difficulties have limited their adoption. Thus, we sought to fabricate a lipid-polymer hybrid that is easily fabricated while maintaining protein function. The hybrid fiber consists of a PCL backbone with conjugated CSS. The hybrid lipid fibers showed improved protein function. In addition, higher lipid concentrations were directly correlated to higher cell capture efficiencies. The third key concept was on the development of dually functionalized lipid fibers and understanding the resulting cell capture efficiencies. Many platforms are unable to simultaneously search for heterogeneous populations of CTCs -- the ability to dually functionalize cell-capturing platforms would address this technological weakness. Studies indicated that dually functionalizing the lipid fibers did not compromise the platforms' abilities to capture the cells of interest. Such dually functionalized fibers allow for a single cell-capture platform to successfully detect heterogeneous populations of CTCs. The body of work encompassed herein describes the use of lipid fibers for antibody immobilization and cell capture. Data from various projects indicate that the use of cholesterol-based fibers produced from electrospun CSS are well suited for protein immobilization. The CSS fibers are able to immobilize equivalent amounts of protein as compared to other immobilization techniques. However, the benefit of these fibers is illustrated by the strong cell-capturing efficiencies, indicating that the immobilized proteins are able to retain their function and selectively target cells of interest. The successful immobilization of proteins and their retained function allows for the development of increasingly sensitive cancer diagnostic tools that are able to screen for CTCs early on in the cancer disease cycle.

A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish

PubMed Central

Xie, Shao-Lin; Bian, Wan-Ping; Wang, Chao; Junaid, Muhammad; Zou, Ji-Xing; Pei, De-Sheng

2016-01-01

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system. PMID:27680290

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Ceramic nanocarriers: versatile nanosystem for protein and peptide delivery.

PubMed

Singh, Deependra; Dubey, Pooja; Pradhan, Madhulika; Singh, Manju Rawat

2013-02-01

Proteins and peptides have been established to be the potential drug candidate for various human diseases. But, delivery of these therapeutic protein and peptides is still a challenge due to their several unfavorable properties. Nanotechnology is expanding as a promising tool for the efficient delivery of proteins and peptides. Among numerous nano-based carriers, ceramic nanoparticles have proven themselves as a unique carrier for protein and peptide delivery as they provide a more stable, bioavailable, readily manufacturable, and acceptable proteins and polypeptide formulation. This article provides an overview of the various aspects of ceramic nanoparticles including their classification, methods of preparation, latest advances, and applications as protein and peptide delivery carriers. Ceramic nanocarriers seem to have potential for preserving structural integrity of proteins and peptides, thereby promoting a better therapeutic effect. This approach thus provides pharmaceutical scientists with a new hope for the delivery of proteins and peptides. Still, considerable study on ceramic nanocarrier is necessary with respect to pharmacokinetics, toxicology, and animal studies to confirm their efficiency as well as safety and to establish their clinical usefulness and scale-up to industrial level.

Endurance exercise training blunts the deleterious effect of high-fat feeding on whole body efficiency

PubMed Central

Holloway, Cameron J.; Murray, Andrew J.; Knight, Nicholas S.; Carter, Emma E.; Kemp, Graham J.; Thompson, Campbell H.; Tyler, Damian J.; Neubauer, Stefan; Robbins, Peter A.; Clarke, Kieran

2011-01-01

We recently showed that a week-long, high-fat diet reduced whole body exercise efficiency in sedentary men by >10% (Edwards LM, Murray AJ, Holloway CJ, Carter EE, Kemp GJ, Codreanu I, Brooker H, Tyler DJ, Robbins PA, Clarke K. FASEB J 25: 1088–1096, 2011). To test if a similar dietary regime would blunt whole body efficiency in endurance-trained men and, as a consequence, hinder aerobic exercise performance, 16 endurance-trained men were given a short-term, high-fat (70% kcal from fat) and a moderate carbohydrate (50% kcal from carbohydrate) diet, in random order. Efficiency was assessed during a standardized exercise task on a cycle ergometer, with aerobic performance assessed during a 1-h time trial and mitochondrial function later measured using 31P-magnetic resonance spectroscopy. The subjects then underwent a 2-wk wash-out period, before the study was repeated with the diets crossed over. Muscle biopsies, for mitochondrial protein analysis, were taken at the start of the study and on the 5th day of each diet. Plasma fatty acids were 60% higher on the high-fat diet compared with moderate carbohydrate diet (P < 0.05). However, there was no change in whole body efficiency and no change in mitochondrial function. Endurance exercise performance was significantly reduced (P < 0.01), most probably due to glycogen depletion. Neither diet led to changes in citrate synthase, ATP synthase, or mitochondrial uncoupling protein 3. We conclude that prior exercise training blunts the deleterious effect of short-term, high-fat feeding on whole body efficiency. PMID:21632846

Activation of IFN-beta element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis.

PubMed Central

Watanabe, N; Sakakibara, J; Hovanessian, A G; Taniguchi, T; Fujita, T

1991-01-01

Expression of the Type I IFN (i.e., IFN-alpha s and IFN-beta) genes is efficiently induced by viruses at the transcriptional level. This induction is mediated by at least two types of positive regulatory elements located in the human IFN-beta gene promoter: (1) the repeated elements which bind both the transcriptional activator IRF-1 and the repressor IRF-2 (IRF-elements; IRF-Es), and (2) the kappa B element (kappa B-E), which binds NF kappa B and is located between the IRF-Es and the TATA box. In this study we demonstrate that a promoter containing synthetic IRF-E, which displays high affinity for the IRFs can be efficiently activated by Newcastle disease virus (NDV). In contrast, such activation was either very weak or nil when cells were treated by IFN-beta or tumor necrosis factor-alpha (TNF-alpha), despite the fact they both efficiently induce de novo synthesis of the short-lived IRF-1 in L929 cells. In fact, efficient activation of the IRF-E apparently requires an event in addition to de novo IRF-1 induction, which can be elicited by NDV even in the presence of protein synthesis inhibitor, cycloheximide. Moreover, efficient activation of the IRF-E by NDV is specifically inhibited by the protein kinase inhibitor, Staurosporin. Hence our results suggest the importance of IRF-1 synthesis and post-translational modification event(s), possibly phosphorylation for the efficient activation of IRF-Es, which are otherwise under negative regulation by IRF-2. Images PMID:1886766

Energy and protein requirements for maintenance of dairy goats during pregnancy and their efficiencies of use.

PubMed

Härter, C J; Lima, L D; Silva, H G O; Castagnino, D S; Rivera, A R; Resende, K T; Teixeira, I A M A

2017-09-01

It has been suggested that maintenance requirements are similar among animals of different physiological stages; however, important physiological changes occur in the maternal body during pregnancy. Therefore, the aim of this study was to determine the energy and protein requirements for the maintenance of pregnant dairy goats and to estimate their efficiency of energy and protein utilization for maintenance and pregnancy. We used 66 multiparous pregnant goats having 49.0 ± 1.59 kg initial BW (around the third or fourth parturition) arranged in a randomized block design with a 3 × 3 factorial scheme including slaughter at different days of pregnancy (DOP; 80, 110, and 140 d) and feed restriction (0, 20, and 40% feed restriction). The comparative slaughter technique was used to estimate energy and protein maintenance requirements. Goats slaughtered at 140 DOP were subjected to digestibility trials at around 80, 110, and 140 DOP to estimate diet metabolizability and N balance (NBAL). Metabolizability decreased with feed restriction and was 63.3 ± 2.16, 55.7 ± 2.35, and 58.2 ± 2.30% at 0, 20, and 40% of feed restriction, respectively ( < 0.01). There was no effect of DOP on NE or the requirements of ME for maintenance (ME), which were 197 and 315 kJ/kg empty body weight (EBW), respectively, and the efficiency of ME utilization for maintenance (k) was 0.63. Similarly, DOP did not affect thedaily net protein requirements for maintenance (NP) estimated using the comparative slaughter technique (1.38 ± 0.512 g/kg EBW; = 0.003) or the NP estimated using NBAL (2.49 ± 0.594 g/kg EBW; < 0.01). The MP requirement for maintenance (MP) estimated using the comparative slaughter technique was not affected by DOP and was 3.22 g MP/kg EBW ( < 0.01). The efficiency of MP utilization for maintenance (k) was 0.43. The efficiency of ME utilization for pregnancy (k) increased with the progress of pregnancy and was 0.058, 0.10, and 0.19 at 80, 110, and 140 DOP, respectively. Similarly, the efficiency of MP utilization for pregnancy (k) increased with DOP and was 0.12, 0.21, and 0.43 at 80, 110, and 140 DOP, respectively. There was no evidence that pregnancy affected NE, ME, NP, and MP or k and k, which were also unaffected by DOP. However, k and k increased with pregnancy progress as a response to the physiological changes that pregnant females are subjected to.

Role of temperature on growth and metabolic rate in the tenebrionid beetles Alphitobius diaperinus and Tenebrio molitor.

PubMed

Bjørge, Julie Dahl; Overgaard, Johannes; Malte, Hans; Gianotten, Natasja; Heckmann, Lars-Henrik

2018-03-10

Insects are increasingly used as a dietary source for food and feed and it is therefore important to understand how rearing conditions affect growth and development of these agricultural animals. Temperature is arguably the most important factor affecting metabolism and growth rate in insects. Here, we investigated how rearing temperature affected growth rate, growth efficiency and macronutrient composition in two species of edible beetle larvae: Alphitobius diaperinus and Tenebrio molitor. Growth rates of both species were quantified at temperatures ranging from 15.2 to 38.0 °C after which we measured protein and lipid content of the different treatment groups. Metabolic rate was measured in a similar temperature range by measuring the rate of O 2 consumption (V·O 2 ) and CO 2 production (V·CO 2 ) using repeated measures closed respirometry. Using these measurements, we calculated the growth efficiency of mealworms by relating the energy assimilation rate to the metabolic rate. Maximum daily growth rates were 18.3% and 16.6% at 31 °C, for A. diaperinus and T. molitor respectively, and we found that A. diaperinus was better at maintaining growth at high temperatures while T. molitor had superior growth at lower temperatures. Both species had highest efficiencies of energy assimilation in the temperature range of 23.3-31.0 °C, with values close to 2 J assimilated/J metabolised in A. diaperinus and around 4 J assimilated/J metabolised in T. molitor. Compared to "conventional" terrestrial livestock, both species of insects were characterised by high growth rates and very high energy conversion efficiency at most experimental temperatures. For A. diaperinus, lipid content was approximately 30% of dry mass and protein content approximately 50% of dry mass across most temperatures. Temperature had a greater influence on the body composition of T. molitor. At 31.0 °C the lipid and protein content was measured to 47.4% and 37.9%, respectively but lipid contents decreased, and protein contents increased when temperatures were higher or lower than 31.0 °C. In summary, rearing temperature had large and independent effects on growth rate, energy assimilation efficiency and protein/lipid content. Accordingly, temperature is a critical parameter to control in commercial insect rearing regardless if the producer wants to optimise production speed, production efficiency or product quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated with Nitrogen Remobilization Efficiency during the Leaf Senescence of Oilseed Rape.

PubMed

Poret, Marine; Chandrasekar, Balakumaran; van der Hoorn, Renier A L; Coquet, Laurent; Jouenne, Thierry; Avice, Jean-Christophe

2017-11-02

Oilseed rape is characterized by a low nitrogen remobilization efficiency during leaf senescence, mainly due to a lack of proteolysis. Because cotyledons are subjected to senescence, it was hypothesized that contrasting protease activities between genotypes may be distinguishable early in the senescence of cotyledons. To verify this assumption, our goals were to (i) characterize protease activities in cotyledons between two genotypes with contrasting nitrogen remobilization efficiency (Ténor and Samouraï) under limiting or ample nitrate supply; and (ii) test the role of salicylic acid (SA) and abscisic acid (ABA) in proteolysis regulation. Protease activities were measured and identified by a proteomics approach combining activity-based protein profiling with LC-MS/MS. As in senescing leaves, chlorophyll and protein contents decrease in senescing cotyledons and are correlated with an increase in serine and cysteine protease activities. Two RD21-like and SAG-12 proteases previously associated with an efficient proteolysis in senescing leaves of Ténor are also detected in senescing cotyledons. The infiltration of ABA and SA provokes the induction of senescence and several cysteine and serine protease activities. The study of protease activities during the senescence of cotyledons seems to be a promising experimental model to investigate the regulation and genotypic variability of proteolysis associated with efficient N remobilization.

Chemical vapor deposition of aminopropyl silanes in microfluidic channels for highly efficient microchip capillary electrophoresis-electrospray ionization-mass spectrometry.

PubMed

Batz, Nicholas G; Mellors, J Scott; Alarie, Jean Pierre; Ramsey, J Michael

2014-04-01

We describe a chemical vapor deposition (CVD) method for the surface modification of glass microfluidic devices designed to perform electrophoretic separations of cationic species. The microfluidic channel surfaces were modified using aminopropyl silane reagents. Coating homogeneity was inferred by precise measurement of the separation efficiency and electroosmotic mobility for multiple microfluidic devices. Devices coated with (3-aminopropyl)di-isopropylethoxysilane (APDIPES) yielded near diffusion-limited separations and exhibited little change in electroosmotic mobility between pH 2.8 and pH 7.5. We further evaluated the temporal stability of both APDIPES and (3-aminopropyl)triethoxysilane (APTES) coatings when stored for a total of 1 week under vacuum at 4 °C or filled with pH 2.8 background electrolyte at room temperature. Measurements of electroosmotic flow (EOF) and separation efficiency during this time confirmed that both coatings were stable under both conditions. Microfluidic devices with a 23 cm long, serpentine electrophoretic separation channel and integrated nanoelectrospray ionization emitter were CVD coated with APDIPES and used for capillary electrophoresis (CE)-electrospray ionization (ESI)-mass spectrometry (MS) of peptides and proteins. Peptide separations were fast and highly efficient, yielding theoretical plate counts over 600,000 and a peak capacity of 64 in less than 90 s. Intact protein separations using these devices yielded Gaussian peak profiles with separation efficiencies between 100,000 and 400,000 theoretical plates.

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System.

PubMed

Bohaciakova, Dasa; Renzova, Tereza; Fedorova, Veronika; Barak, Martin; Kunova Bosakova, Michaela; Hampl, Ales; Cajanek, Lukas

2017-11-01

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

PubMed

Siapati, Elena K; Bigger, Brian W; Miskin, James; Chipchase, Daniel; Parsley, Kathryn L; Mitrophanous, Kyriacos; Themis, Mike; Thrasher, Adrian J; Bonnet, Dominique

2005-09-01

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

PubMed Central

Jackson, Kasey L.; Dayton, Robert D.; Deverman, Benjamin E.; Klein, Ronald L.

2016-01-01

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats. PMID:27867348

Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

PubMed

Jackson, Kasey L; Dayton, Robert D; Deverman, Benjamin E; Klein, Ronald L

2016-01-01

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

Polyethyleneimine grafted short halloysite nanotubes for gene delivery.

PubMed

Long, Zheru; Zhang, Jun; Shen, Yan; Zhou, Changren; Liu, Mingxian

2017-12-01

Inorganic nanoparticles have attracted much attentions in gene delivery because of their desirable characteristics including low toxicity, well-controlled characteristics, high gene delivery efficiency, and multi-functionalities. Here, natural occurred halloysite nanotubes (HNTs) were developed as a novel non-viral gene vector. To increase the efficiency of endocytosis, HNTs were firstly shortened into an appropriate size (~200nm). Then polyethyleneimine (PEI) was grafted onto HNTs to bind green fluorescence protein (GFP) labeled pDNA. The structure and physical-chemical properties of PEI grafted HNTs (PEI-g-HNTs) were characterized by various methods. PEI-g-HNTs show lower cytotoxicity than PEI. PEI-g-HNTs are positively charged and can bind DNA tightly at designed N/P ratio from 5:1 to 40:1. PEI-g-HNTs/pDNA complexes show much higher transfection efficiency towards both 293T and HeLa cells compared with PEI/pDNA complexes at the equivalent N/P ratio. The transfection efficiencies of PEI-g-HNTs/pDNA complex towards HeLa cell can reach to 44.4% at N/P ratio of 20. PEI-g-HNTs/pDNA complexes possess a higher GFP protein expression than PEI/pDNA from simple western immunoblots. So, PEI-g-HNTs are potential gene vectors with good biocompatibility and high transfection efficiency, which have promising applications in cancer gene therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Performance efficiency of feed utilization, relative growth rate, and survival rate of common carp (Cyprinus carpio) through the addition of phytase in the feed

NASA Astrophysics Data System (ADS)

Rachmawati, D.; Samidjan, I.

2018-04-01

The purpose of this study was to determine the effect of adding phytase enzyme in the feed on digestibility of feed, efficiency of feed utilization, relative growth rate and survival rate of Common carp (Cyprinus carpio). Fish samples in this research were Common carp with an average - weight of 3.34 ± 0,16 g/fish. The treatments were adding the phytase enzyme in the feed with the different level of doses. Those were A (0 U kg-1 feed), B (500 U kg-1 feed), C (1.000 U kg-1 feed g) and D (1.500 U kg-1 feed). Observation was conducted on digestibility of protein (ADCP), digestibility of phosphor (ADCF), efficiency of feed utilization (EFU), relative growth rate (RGR), protein efficiency ratio (PER), feed conversion ratio (FCR), survival rate (SR) and water quality parameters. The results show that the addition of phytase enzyme significantly (P<0.01) affected on ADCP, ADCF, EFU, RGR, FCR, and PER, on the other hand it insignificantly (P>0.05) affected on SR of common carp. Based on results, it was concluded that optimum doses of phytase enzyme feed in terms of digestibility of feed, efficiency utilization of Feed and growth rate of Common carp ranges from 943 to 1100 U kg-1 feed

A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

PubMed

Salomone, Fabrizio; Cardarelli, Francesco; Di Luca, Mariagrazia; Boccardi, Claudia; Nifosì, Riccardo; Bardi, Giuseppe; Di Bari, Lorenzo; Serresi, Michela; Beltram, Fabio

2012-11-10

Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched. In this context, antimicrobial peptides (AMPs) provide viable templates for the design of new membrane-disruptive motifs. In particular the Cecropin-A and Melittin hybrids (CMs) are among the smallest and most effective peptides with membrane-perturbing abilities. Here we present a novel chimeric peptide in which the Tat(11) motif is fused to the CM(18) hybrid (KWKLFKKIGAVLKVLTTG, residues 1-7 of Cecropin-A and 2-12 of Melittin). When administered to cells, CM(18)-Tat(11) combines the two desired functionalities: efficient uptake and destabilization of endocytotic-vesicle membranes. We show that this chimeric peptide effectively increases cargo-molecule cytoplasm availability and allows the subsequent intracellular localization of diverse membrane-impermeable molecules (i.e. Tat(11)-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with no detectable cytotoxicity. The present results open the way to the rational engineering of "modular" cell-penetrating peptides (CPPs) that combine (i) efficient translocation from the extracellular milieu into vesicles and (ii) efficient release of molecules from vesicles into the cytoplasm. Copyright © 2012 Elsevier B.V. All rights reserved.

Recent Developments and Applications of the MMPBSA Method

PubMed Central

Wang, Changhao; Greene, D'Artagnan; Xiao, Li; Qi, Ruxi; Luo, Ray

2018-01-01

The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach has been widely applied as an efficient and reliable free energy simulation method to model molecular recognition, such as for protein-ligand binding interactions. In this review, we focus on recent developments and applications of the MMPBSA method. The methodology review covers solvation terms, the entropy term, extensions to membrane proteins and high-speed screening, and new automation toolkits. Recent applications in various important biomedical and chemical fields are also reviewed. We conclude with a few future directions aimed at making MMPBSA a more robust and efficient method. PMID:29367919

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

PubMed

Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

2010-10-01

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular engineering of industrial enzymes: recent advances and future prospects.

PubMed

Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

2014-01-01

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

Protein extraction into the bicontinuous microemulsion phase of a Water/SDS/pentanol/dodecane winsor-III system: Effect on nanostructure and protein conformation

DOE PAGES

Hayes, Douglas G.; Ye, Ran; Dunlap, Rachel N.; ...

2017-09-07

Bicontinuous microemulsions (BμEs), consisting of water and oil nanodomains separated by surfactant monolayers of near-zero curvature, are potentially valuable systems for purification and delivery of biomolecules, for hosting multiphasic biochemical reactions, and as templating media for preparing nanomaterials. We formed Winsor-III systems by mixing aqueous protein and sodium dodecyl sulfate (SDS) solutions with dodecane and 1-pentanol (cosurfactant) to efficiently extract proteins into the middle (BμE) phase. Bovine serum albumin (BSA) and cytochrome c partitioned to the BμE phase at 64% and 81% efficiency, respectively, producing highly concentrated protein solutions (32 and 44 g L –1, respectively), through release of watermore » and oil from the BμEs. Circular dichroism spectroscopic analysis demonstrated that BSA underwent minor secondary structural changes upon incorporation into BμEs, while the secondary structure of cytochrome c and pepsin underwent major changes. Small-angle x-ray scattering (SAXS) results show that proteins promoted an increase of the interfacial fluidity and surface area per volume for the BμE surfactant monolayers, and that each protein uniquely altered self-assembly in the Winsor-III systems. Cytochrome c partitioned via electrostatic attractions between SDS and the protein’s positively-charged groups, residing near the surfactant head groups of BμE monolayers, where it decreased surfactant packing efficiency. BSA partitioned through formation of SDS-BSA complexes via hydrophobic and electrostatic attractive interactions. As the BSA-SDS ratio increased, complexes’ partitioning favored BμEs over the oil excess phase due to the increased hydrophilicity of the complexes. In conclusion, this study demonstrates the potential utility of BμEs to purify proteins and prepare nanostructured fluids possessing high protein concentration.« less

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

PubMed

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Protein extraction into the bicontinuous microemulsion phase of a Water/SDS/pentanol/dodecane winsor-III system: Effect on nanostructure and protein conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayes, Douglas G.; Ye, Ran; Dunlap, Rachel N.

Bicontinuous microemulsions (BμEs), consisting of water and oil nanodomains separated by surfactant monolayers of near-zero curvature, are potentially valuable systems for purification and delivery of biomolecules, for hosting multiphasic biochemical reactions, and as templating media for preparing nanomaterials. We formed Winsor-III systems by mixing aqueous protein and sodium dodecyl sulfate (SDS) solutions with dodecane and 1-pentanol (cosurfactant) to efficiently extract proteins into the middle (BμE) phase. Bovine serum albumin (BSA) and cytochrome c partitioned to the BμE phase at 64% and 81% efficiency, respectively, producing highly concentrated protein solutions (32 and 44 g L –1, respectively), through release of watermore » and oil from the BμEs. Circular dichroism spectroscopic analysis demonstrated that BSA underwent minor secondary structural changes upon incorporation into BμEs, while the secondary structure of cytochrome c and pepsin underwent major changes. Small-angle x-ray scattering (SAXS) results show that proteins promoted an increase of the interfacial fluidity and surface area per volume for the BμE surfactant monolayers, and that each protein uniquely altered self-assembly in the Winsor-III systems. Cytochrome c partitioned via electrostatic attractions between SDS and the protein’s positively-charged groups, residing near the surfactant head groups of BμE monolayers, where it decreased surfactant packing efficiency. BSA partitioned through formation of SDS-BSA complexes via hydrophobic and electrostatic attractive interactions. As the BSA-SDS ratio increased, complexes’ partitioning favored BμEs over the oil excess phase due to the increased hydrophilicity of the complexes. In conclusion, this study demonstrates the potential utility of BμEs to purify proteins and prepare nanostructured fluids possessing high protein concentration.« less

The effect of silicon on the leaf proteome of rice (Oryza sativa L.) plants under cadmium-stress.

PubMed

Nwugo, Chika C; Huerta, Alfredo J

2011-02-04

The best known silicon (Si)-accumulating plant, rice (Oryza sativa L.), stores most of its Si in leaves, but the importance of Si has been limited to a mechanical role. Our initial studies showed that Si-induced cadmium (Cd) tolerance is mediated by the enhancement of instantaneous water-use-efficiency, carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), and light-use-efficiency in leaves of rice plants. In this study, we investigated changes in the rice leaf proteome in order to identify molecular mechanisms involved in Si-induced Cd tolerance. Our study identified 60 protein spots that were differentially regulated due to Cd and/or Si treatments. Among them, 50 were significantly regulated by Si, including proteins associated with photosynthesis, redox homeostasis, regulation/protein synthesis, pathogen response and chaperone activity. Interestingly, we observed a Si-induced up-regulation of a class III peroxidase and a thaumatin-like protein irrespective of Cd treatment, in addition to a Cd-induced up-regulation of protein disulfide isomerase, a HSP70 homologue, a NADH-ubiquinone oxidoreductase, and a putative phosphogluconate dehydrogenase, especially in the presence of Si. Taken together, our study sheds light on molecular mechanisms involved in Si-induced Cd tolerance in rice leaves and suggests a more active involvement of Si in plant physiological processes than previously proposed.

Oral Vaccination with a DNA Vaccine Encoding Capsid Protein of Duck Tembusu Virus Induces Protection Immunity

PubMed Central

Shen, Haoyue; Jia, Renyong; Wang, Mingshu; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Zhao, Xinxin; Yang, Qiao; Wu, Ying; Liu, Yunya; Zhang, Ling; Yin, Zhongqiong; Jing, Bo

2018-01-01

The emergence of duck tembusu virus (DTMUV), a new member of the Flavivirus genus, has caused great economical loss in the poultry industry in China. Since the outbreak and spread of DTMUV is hard to control in a clinical setting, an efficient and low-cost oral delivery DNA vaccine SL7207 (pVAX1-C) based on the capsid protein of DTMUV was developed and evaluated in this study. The antigen capsid protein was expressed from the DNA vaccine SL7207 (pVAX1-C), both in vitro and in vivo. The humoral and cellular immune responses in vivo were observed after oral immunization with the SL7207 (pVAX1-C) DNA vaccine. High titers of the specific antibody against the capsid protein and the neutralizing antibody against the DTMUV virus were both detected after inoculation. The ducks were efficiently protected from lethal DTMUV exposure by the SL7207 (pVAX1-C) vaccine in this experiment. Taken together, we demonstrated that the capsid protein of DTMUV possesses a strong immunogenicity against the DTMUV infection. Moreover, an oral delivery of the DNA vaccine SL7207 (pVAX1-C) utilizing Salmonella SL7207 was an efficient way to protect the ducks against DTMUV infection and provides an economic and fast vaccine delivery strategy for a large scale clinical use. PMID:29642401

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context.

PubMed

Lukes, Julius; Paris, Zdenek; Regmi, Sandesh; Breitling, Reinhard; Mureev, Sergey; Kushnir, Susanna; Pyatkov, Konstantin; Jirků, Milan; Alexandrov, Kirill A

2006-08-01

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

A Comparative Study of Proteolytic Mechanisms during Leaf Senescence of Four Genotypes of Winter Oilseed Rape Highlighted Relevant Physiological and Molecular Traits for NRE Improvement

PubMed Central

Girondé, Alexandra; Poret, Marine; Etienne, Philippe; Trouverie, Jacques; Bouchereau, Alain; Le Cahérec, Françoise; Leport, Laurent; Niogret, Marie-Françoise; Avice, Jean-Christophe

2015-01-01

Winter oilseed rape is characterized by a low N use efficiency related to a weak leaf N remobilization efficiency (NRE) at vegetative stages. By investigating the natural genotypic variability of leaf NRE, our goal was to characterize the relevant physiological traits and the main protease classes associated with an efficient proteolysis and high leaf NRE in response to ample or restricted nitrate supply. The degradation rate of soluble proteins and D1 protein (a thylakoid-bound protein) were correlated to N remobilization, except for the genotype Samouraï which showed a low NRE despite high levels of proteolysis. Under restricted nitrate conditions, high levels of soluble protein degradation were associated with serine, cysteine and aspartic proteases at acidic pH. Low leaf NRE was related to a weak proteolysis of both soluble and thylakoid-bound proteins. The results obtained on the genotype Samouraï suggest that the timing between the onset of proteolysis and abscission could be a determinant. The specific involvement of acidic proteases suggests that autophagy and/or senescence-associated vacuoles are implicated in N remobilization under low N conditions. The data revealed that the rate of D1 degradation could be a relevant indicator of leaf NRE and might be used as a tool for plant breeding. PMID:27135221

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

PubMed

Röhm, Martina; Carle, Stefan; Maigler, Frank; Flamm, Johannes; Kramer, Viktoria; Mavoungou, Chrystelle; Schmid, Otmar; Schindowski, Katharina

2017-10-30

Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

3Drefine: an interactive web server for efficient protein structure refinement.

PubMed

Bhattacharya, Debswapna; Nowotny, Jackson; Cao, Renzhi; Cheng, Jianlin

2016-07-08

3Drefine is an interactive web server for consistent and computationally efficient protein structure refinement with the capability to perform web-based statistical and visual analysis. The 3Drefine refinement protocol utilizes iterative optimization of hydrogen bonding network combined with atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields for efficient protein structure refinement. The method has been extensively evaluated on blind CASP experiments as well as on large-scale and diverse benchmark datasets and exhibits consistent improvement over the initial structure in both global and local structural quality measures. The 3Drefine web server allows for convenient protein structure refinement through a text or file input submission, email notification, provided example submission and is freely available without any registration requirement. The server also provides comprehensive analysis of submissions through various energy and statistical feedback and interactive visualization of multiple refined models through the JSmol applet that is equipped with numerous protein model analysis tools. The web server has been extensively tested and used by many users. As a result, the 3Drefine web server conveniently provides a useful tool easily accessible to the community. The 3Drefine web server has been made publicly available at the URL: http://sysbio.rnet.missouri.edu/3Drefine/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Co-adaption of tRNA gene copy number and amino acid usage influences translation rates in three life domains

PubMed Central

Du, Meng-Ze; Wei, Wen; Qin, Lei; Liu, Shuo; Zhang, An-Ying; Zhang, Yong; Zhou, Hong

2017-01-01

Abstract Although more and more entangled participants of translation process were realized, how they cooperate and co-determine the final translation efficiency still lacks details. Here, we reasoned that the basic translation components, tRNAs and amino acids should be consistent to maximize the efficiency and minimize the cost. We firstly revealed that 310 out of 410 investigated genomes of three domains had significant co-adaptions between the tRNA gene copy numbers and amino acid compositions, indicating that maximum efficiency constitutes ubiquitous selection pressure on protein translation. Furthermore, fast-growing and larger bacteria are found to have significantly better co-adaption and confirmed the effect of this pressure. Within organism, highly expressed proteins and those connected to acute responses have higher co-adaption intensity. Thus, the better co-adaption probably speeds up the growing of cells through accelerating the translation of special proteins. Experimentally, manipulating the tRNA gene copy number to optimize co-adaption between enhanced green fluorescent protein (EGFP) and tRNA gene set of Escherichia coli indeed lifted the translation rate (speed). Finally, as a newly confirmed translation rate regulating mechanism, the co-adaption reflecting translation rate not only deepens our understanding on translation process but also provides an easy and practicable method to improve protein translation rates and productivity. PMID:28992099

Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

PubMed Central

Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J.; Gurdon, John B.; Jullien, Jerome

2014-01-01

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development. PMID:25244019

Brd4 Is Required for E2-Mediated Transcriptional Activation but Not Genome Partitioning of All Papillomaviruses†

PubMed Central

McPhillips, M. G.; Oliveira, J. G.; Spindler, J. E.; Mitra, R.; McBride, A. A.

2006-01-01

Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes. PMID:16973557

Enhancing Sensitivity of Liquid Chromatography-Mass Spectrometry of Peptides and Proteins Using Supercharging Agents.

PubMed

Nshanian, Michael; Lakshmanan, Rajeswari; Chen, Hao; Ogorzalek Loo, Rachel R; Loo, Joseph A

2018-04-01

Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

Preparation and characterization of protein-loaded poly(epsilon-caprolactone) microparticles for oral vaccine delivery.

PubMed

Benoit, M A; Baras, B; Gillard, J

1999-07-05

This paper describes the conditions of preparation of poly(epsilon-caprolactone) (PCL) microparticles with a mean size between 5 and 10 microm, obtained by a double emulsion-solvent evaporation technique, suitable for oral vaccine delivery. Bovine serum albumin (BSA) was used as water-soluble model antigen for encapsulation. Different parameters influencing the microparticle size, the BSA loading and entrapment efficiency were investigated. Spherical, smooth and homogeneously distributed microparticles were produced with a BSA loading and entrapment efficiency reaching, respectively, 5% (w/w) and 30%. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) analyses of BSA released from these particles confirmed that the entrapped protein seemed to remain unaltered by the protein encapsulation process. Copyright.

One-step purification of assembly-competent tubulin from diverse eukaryotic sources

PubMed Central

Widlund, Per O.; Podolski, Marija; Reber, Simone; Alper, Joshua; Storch, Marko; Hyman, Anthony A.; Howard, Jonathon; Drechsel, David N.

2012-01-01

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. PMID:22993214

Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

PubMed

Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A

2011-04-28

Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

USDA-ARS?s Scientific Manuscript database

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or an...

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

PubMed

Subrahmanyam, S; Cronan, J E

1999-01-21

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

PubMed

Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

2013-04-28

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of divalent ions on the optical emission behavior of protein thin films

NASA Astrophysics Data System (ADS)

Bhowal, Ashim Chandra; Kundu, Sarathi

2016-05-01

Photoluminescence behaviors of proteinthin film, bovine serum albumin (BSA) have been studied in the presence of three divalent ions (Mg2+, Ca2+ and Ba2+) at different temperatures using fluorescence spectroscopy. Film thickness and morphology have been studied using atomic force microscopy. Variation of different physicochemical parameters like temperature, solvent polarity, pH, ionic strength, substrate binding etc. can make conformational changes in the protein structure and hence influences the emission behavior.In thin film conformation of BSA, dynamic quenching behavior has beenidentified in the presence of all the three divalent ions at pH≈ 5.5. Depending upon the charge density of the divalent ions interaction with protein molecules modifies and as a result quenching efficiency varies. Also after heat treatment, conformation of the protein molecules changes and as a result the quenching efficiency enhances than that of the unheated films. Studies on such protein-ion interactions and conformational variation may explore various functions of protein when it will adsorb on soft surfaces like membranes, vesicles, etc.

Removal of detergents from proteins and peptides in a spin-column format.

PubMed

Antharavally, Babu S

2012-08-01

To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. This unit describes the use of a high-performance resin that offers exceptional detergent removal for proteins and peptides. The easy-to-use spin format significantly improves results over the standard drip column and batch methodologies, with >95% removal of 1% to 5% detergents, including SDS, sodium deoxycholate, CHAPS, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Detergent removal efficiency is evaluated using colorimetric methods and mass spectrometry (MS). BSA tryptic peptides have been successfully analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption/ionization (MALDI)-MS for identification of protein, following detergent removal using the resin. Advantages of this method include speed (less than 15 min), efficient detergent removal, and high recovery of proteins and peptides. © 2012 by John Wiley & Sons, Inc.

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

PubMed

Sato, Yoshimi; Kojima, Rieko; Okumura, Masaki; Hagiwara, Masatoshi; Masui, Shoji; Maegawa, Ken-ichi; Saiki, Masatoshi; Horibe, Tomohisa; Suzuki, Mamoru; Inaba, Kenji

2013-01-01

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

PubMed

Seo, Ki-Weon; Kim, Dong-Heon; Kim, Ah Hyun; Yoo, Han-Sang; Lee, Kyung-Yeol; Jang, Yong-Suk

2011-01-01

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency.

PubMed

Rudhe, Charlotta; Clifton, Rachel; Whelan, James; Glaser, Elzbieta

2002-12-06

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.

Novel value-added uses for sweet potato juice and flour in polyphenol- and protein-enriched functional food ingredients

PubMed Central

Grace, Mary H; Truong, An N; Truong, Van-Den; Raskin, Ilya; Lila, Mary Ann

2015-01-01

Blackcurrant, blueberry, and muscadine grape juices were efficiently sorbed, concentrated, and stabilized into dry granular ingredient matrices which combined anti-inflammatory and antioxidant fruit polyphenols with sweet potato functional constituents (carotenoids, vitamins, polyphenols, fibers). Total phenolics were highest in blackcurrant-orange sweet potato ingredient matrices (34.03 mg/g), and lowest in muscadine grape-yellow sweet potato matrices (10.56 mg/g). Similarly, anthocyanins were most concentrated in blackcurrant-fortified orange and yellow sweet potato matrices (5.40 and 6.54 mg/g, respectively). Alternatively, other protein-rich edible matrices (defatted soy flour, light roasted peanut flour, and rice protein concentrate) efficiently captured polyphenols (6.09–9.46 mg/g) and anthocyanins (0.77–1.27 mg/g) from purple-fleshed sweet potato juice, with comparable efficiency. Antioxidant activity correlated well with total phenolic content. All formulated ingredient matrices stabilized and preserved polyphenols for up to 24 weeks, even when stored at 37°C. Complexation with juice-derived polyphenols did not significantly alter protein or carbohydrate profiles of the matrices. Sensory evaluation of the ingredient matrices suggested potential uses for a wide range of functional food products. PMID:26405527

Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation.

PubMed

Deng, Zhongyuan; Zhang, Shen; Gu, Shaohua; Ni, Xinzhi; Zeng, Wenxian; Li, Xianchun

2018-01-17

The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis -acting elements, trans -acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo.

PubMed

Cheng, Xu; Qin, Jiejie; Wang, Xin; Zha, Qian; Yao, Weijing; Fu, Shengxiang; Tang, Rupei

2018-05-03

It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid-degradable nanogels were prepared by cross-linking methacrylated soy protein with an acid-labile ortho ester cross-linker (NG1), and then modified with lactobionic acid (LA) to give tumor-targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH-triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA-modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over-expressed HepG2 cells. In vivo antitumor experimentproved that LA modification could significantly enhance the tumor-targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein-based nanogels did not cause any damage to normal organs. Overall, these pH-sensitive and tumor-targeting soy protein-based nanogels can be potential drug carriers for efficient tumor treatment. Copyright © 2018. Published by Elsevier B.V.

Coupling of metal-organic frameworks-containing monolithic capillary-based selective enrichment with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for efficient analysis of protein phosphorylation.

PubMed

Li, Daojin; Yin, Danyang; Chen, Yang; Liu, Zhen

2017-05-19

Protein phosphorylation is a major post-translational modification, which plays a vital role in cellular signaling of numerous biological processes. Mass spectrometry (MS) has been an essential tool for the analysis of protein phosphorylation, for which it is a key step to selectively enrich phosphopeptides from complex biological samples. In this study, metal-organic frameworks (MOFs)-based monolithic capillary has been successfully prepared as an effective sorbent for the selective enrichment of phosphopeptides and has been off-line coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for efficient analysis of phosphopeptides. Using š-casein as a representative phosphoprotein, efficient phosphorylation analysis by this off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also demonstrated. Through introducing large surface areas and highly ordered pores of MOFs into monolithic column, the MOFs-based monolithic capillary exhibited several significant advantages, such as excellent selectivity toward phosphopeptides, superb tolerance to interference and simple operation procedure. Because of these highly desirable properties, the MOFs-based monolithic capillary could be a useful tool for protein phosphorylation analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

PubMed Central

2010-01-01

Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory. PMID:20836848

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.

PubMed

Hatahet, Feras; Nguyen, Van Dat; Salo, Kirsi E H; Ruddock, Lloyd W

2010-09-13

The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

PubMed

Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

2017-01-01

In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient protein structure search using indexing methods

PubMed Central

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively. PMID:23691543

Efficient protein structure search using indexing methods.

PubMed

Kim, Sungchul; Sael, Lee; Yu, Hwanjo

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively.

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

PubMed Central

Python, Sylvie; Gerber, Markus; Suter, Rolf; Ruggli, Nicolas; Summerfield, Artur

2013-01-01

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic. PMID:23785283

A Novel Approach To Improve the Efficiency of Block Freeze Concentration Using Ice Nucleation Proteins with Altered Ice Morphology.

PubMed

Jin, Jue; Yurkow, Edward J; Adler, Derek; Lee, Tung-Ching

2017-03-22

Freeze concentration is a separation process with high success in product quality. The remaining challenge is to achieve high efficiency with low cost. This study aims to evaluate the potential of using ice nucleation proteins (INPs) as an effective method to improve the efficiency of block freeze concentration while also exploring the related mechanism of ice morphology. Our results show that INPs are able to significantly improve the efficiency of block freeze concentration in a desalination model. Using this experimental system, we estimate that approximately 50% of the energy cost can be saved by the inclusion of INPs in desalination cycles while still meeting the EPA standard of drinking water (<500 ppm). Our investigative tools for ice morphology include optical microscopy and X-ray computed tomography imaging analysis. Their use indicates that INPs promote the development of a lamellar structured ice matrix with larger hydraulic diameters, which facilitates brine drainage and contains less brine entrapment as compared to control samples. These results suggest great potential for applying INPs to develop an energy-saving freeze concentration method via the alteration of ice morphology.

Non-canonical mechanism for translational control in bacteria: synthesis of ribosomal protein S1

PubMed Central

Boni, Irina V.; Artamonova, Valentina S.; Tzareva, Nina V.; Dreyfus, Marc

2001-01-01

Translation initiation region (TIR) of the rpsA mRNA encoding ribosomal protein S1 is one of the most efficient in Escherichia coli despite the absence of a canonical Shine–Dalgarno-element. Its high efficiency is under strong negative autogenous control, a puzzling phenomenon as S1 has no strict sequence specificity. To define sequence and structural elements responsible for translational efficiency and autoregulation of the rpsA mRNA, a series of rpsA′–′lacZ chromosomal fusions bearing various mutations in the rpsA TIR was created and tested for β-galactosidase activity in the absence and presence of excess S1. These in vivo results, as well as data obtained by in vitro techniques and phylogenetic comparison, allow us to propose a model for the structural and functional organization of the rpsA TIR specific for proteobacteria related to E.coli. According to the model, the high efficiency of translation initiation is provided by a specific fold of the rpsA leader forming a non-contiguous ribosome entry site, which is destroyed upon binding of free S1 when it acts as an autogenous repressor. PMID:11483525

Mass spectrometry compatible surfactant for optimized in-gel protein digestion.

PubMed

Saveliev, Sergei V; Woodroofe, Carolyn C; Sabat, Grzegorz; Adams, Christopher M; Klaubert, Dieter; Wood, Keith; Urh, Marjeta

2013-01-15

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.

Micro-emulsification/encapsulation of krill oil by complex coacervation with krill protein isolated using isoelectric solubilization/precipitation.

PubMed

Shi, Liu; Beamer, Sarah K; Yang, Hong; Jaczynski, Jacek

2018-04-01

This study determined feasibility of krill protein isolated with isoelectric solubilization/precipitation (ISP) as wall material to microencapsulate krill oil by freeze-drying. Effects of krill oil/krill protein ratio on properties of microcapsules were investigated. With increased ratio, crude protein of microcapsules decreased, while total lipid increased. Although microcapsule oil loading capacity increased, loading and encapsulation efficiencies decreased. Thin layer chromatography (TLC) confirmed abundance of phospholipids, which are amphiphilic; and thus, resulted in stable emulsion (emulsion stability index). Microcapsules contained ω-3 polyunsaturated fatty acids (PUFAs) at 43-60, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at 28-41 and 9-11 g/100g of total FAs, respectively. SDS-PAGE electrophoresis revealed proteolysis of ISP krill protein, probably causing reduced loading and encapsulation efficiencies. SEM showed that krill oil/krill protein ratio affected surface microstructure. ISP krill protein showed potential as a wall material to microencapsulate krill oil; and thus, expand application of krill oil/protein for human consumption. Copyright © 2017 Elsevier Ltd. All rights reserved.

A combinatorial perspective of the protein inference problem.

PubMed

Yang, Chao; He, Zengyou; Yu, Weichuan

2013-01-01

In a shotgun proteomics experiment, proteins are the most biologically meaningful output. The success of proteomics studies depends on the ability to accurately and efficiently identify proteins. Many methods have been proposed to facilitate the identification of proteins from peptide identification results. However, the relationship between protein identification and peptide identification has not been thoroughly explained before. In this paper, we devote ourselves to a combinatorial perspective of the protein inference problem. We employ combinatorial mathematics to calculate the conditional protein probabilities (protein probability means the probability that a protein is correctly identified) under three assumptions, which lead to a lower bound, an upper bound, and an empirical estimation of protein probabilities, respectively. The combinatorial perspective enables us to obtain an analytical expression for protein inference. Our method achieves comparable results with ProteinProphet in a more efficient manner in experiments on two data sets of standard protein mixtures and two data sets of real samples. Based on our model, we study the impact of unique peptides and degenerate peptides (degenerate peptides are peptides shared by at least two proteins) on protein probabilities. Meanwhile, we also study the relationship between our model and ProteinProphet. We name our program ProteinInfer. Its Java source code, our supplementary document and experimental results are available at: >http://bioinformatics.ust.hk/proteininfer.

Proteomic analyses of the environmental toxicity of carcinogenic chemicals

EPA Science Inventory

Protein expression and posttranslational modifications consistently change in response to the exposure to environmental chemicals. Recent technological advances in proteomics provide new tools for more efficient characterization of protein expression and posttranslational modific...

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

PubMed

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus.

PubMed

Sharma Ghimire, Prakriti; Ouyang, Haomiao; Wang, Qian; Luo, Yuanming; Shi, Bo; Yang, Jinghua; Lü, Yang; Jin, Cheng

2016-12-02

Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

An intramolecular disulfide bridge between Cys-7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus.

PubMed Central

Nassal, M; Rieger, A

1993-01-01

Hepatitis B virus, the prototypic member of the Hepadnaviridae, is a small enveloped DNA virus that replicates via reverse transcription. Efficient usage of its compact 3.2-kb genome is exemplified by the pre-C/C gene from which two proteins with largely overlapping primary sequences but distinctly different properties are synthesized: the self-assembling core protein p21c (hepatitis B core antigen [HbcAg]) and the secretory, nonparticulate protein p17e (hepatitis B e antigen [HbeAg]). Mature p17e carries a 10-amino-acid N-terminal extension with a Cys residue (Cys-7). Using transient transfection of a human liver cell line with constructs expressing wild-type p17 or a series of Cys mutants of p17, we show that Cys-7 forms an intramolecular S-S bond to Cys61, which in assembly-competent core proteins is available for intermolecular disulfide bonds between two neighboring subunits. Removal of the Cys-7/Cys61 bond by mutating either residue has differential effects: in the absence of Cys-7, secretion is relatively efficient and independent of Cys61; however, the molecules are exported as homodimers exhibiting both HBe and HBc antigenicity. In the absence of Cys61, the nonpaired Cys-7 interferes with secretion efficiency. The amino acid sequence flanking Cys-7 also contributes to the formation of the proper intramolecular S-S bond. These results suggest that the Cys-7/Cys61 bond imposes on p17e a conformation that is critical for its secretion and distinct biophysical and antigenic properties. This mechanism adds selective disulfide formation to the repertoire of hepatitis B virus for efficient use of its tiny genome. Images PMID:8510224

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

PubMed

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

PubMed Central

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

PubMed

Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

2015-12-01

Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

PubMed

Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

2015-02-01

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium. © 2014 Wiley Periodicals, Inc.

Design and function of biomimetic multilayer water purification membranes

PubMed Central

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L.; Buehler, Markus J.

2017-01-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences. PMID:28435877

Design and function of biomimetic multilayer water purification membranes.

PubMed

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L; Buehler, Markus J

2017-04-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences.

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect.

PubMed

Montoliu-Gaya, Laia; Esquerda-Canals, Gisela; Bronsoms, Silvia; Villegas, Sandra

2017-01-01

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer's Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

PubMed Central

Montoliu-Gaya, Laia; Esquerda-Canals, Gisela; Bronsoms, Silvia

2017-01-01

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer’s Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety. PMID:28771492

Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

PubMed

Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

2013-02-01

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs. Copyright © 2012 Elsevier B.V. All rights reserved.

Protein structural similarity search by Ramachandran codes

PubMed Central

Lo, Wei-Cheng; Huang, Po-Jung; Chang, Chih-Hung; Lyu, Ping-Chiang

2007-01-01

Background Protein structural data has increased exponentially, such that fast and accurate tools are necessary to access structure similarity search. To improve the search speed, several methods have been designed to reduce three-dimensional protein structures to one-dimensional text strings that are then analyzed by traditional sequence alignment methods; however, the accuracy is usually sacrificed and the speed is still unable to match sequence similarity search tools. Here, we aimed to improve the linear encoding methodology and develop efficient search tools that can rapidly retrieve structural homologs from large protein databases. Results We propose a new linear encoding method, SARST (Structural similarity search Aided by Ramachandran Sequential Transformation). SARST transforms protein structures into text strings through a Ramachandran map organized by nearest-neighbor clustering and uses a regenerative approach to produce substitution matrices. Then, classical sequence similarity search methods can be applied to the structural similarity search. Its accuracy is similar to Combinatorial Extension (CE) and works over 243,000 times faster, searching 34,000 proteins in 0.34 sec with a 3.2-GHz CPU. SARST provides statistically meaningful expectation values to assess the retrieved information. It has been implemented into a web service and a stand-alone Java program that is able to run on many different platforms. Conclusion As a database search method, SARST can rapidly distinguish high from low similarities and efficiently retrieve homologous structures. It demonstrates that the easily accessible linear encoding methodology has the potential to serve as a foundation for efficient protein structural similarity search tools. These search tools are supposed applicable to automated and high-throughput functional annotations or predictions for the ever increasing number of published protein structures in this post-genomic era. PMID:17716377

The efficiency of contact lens care regimens on protein removal from hydrogel and silicone hydrogel lenses

PubMed Central

Heynen, Miriam; Liu, Lina; Sheardown, Heather; Jones, Lyndon

2010-01-01

Purpose To investigate the efficiency of lysozyme and albumin removal from silicone hydrogel and conventional contact lenses, using a polyhexamethylene biguanide multipurpose solution (MPS) in a soaking or rubbing/soaking application and a hydrogen peroxide system (H2O2). Methods Etafilcon A, lotrafilcon B and balafilcon A materials were incubated in protein solutions for up to 14 days. Lenses were either placed in radiolabeled protein to quantify the amount deposited or in fluorescent-conjugated protein to identify its location, using confocal laser scanning microscopy (CLSM). Lenses were either rinsed with PBS or soaked overnight in H2O2 or MPS with and without lens rubbing. Results After 14 days lysozyme was highest on etafilcon A (2,200 μg) >balafilcon A (50 µg) >lotrafilcon B (9.7 µg) and albumin was highest on balafilcon A (1.9 µg) =lotrafilcon B (1.8 µg) >etafilcon A (0.2 µg). Lysozyme removal was greatest for balafilcon A >etafilcon A >lotrafilcon B, with etafilcon A showing the most change in protein distribution. Albumin removal was highest from etafilcon A >balafilcon A >lotrafilcon B. H2O2 exhibited greater lysozyme removal from etafilcon A compared to both MPS procedures (p<0.001) but performed similarly for lotrafilcon B and balafilcon A lenses (p>0.62). Albumin removal was solely material specific, while all care regimens performed to a similar degree (p>0.69). Conclusions Protein removal efficiency for the regimens evaluated depended on the lens material and protein type. Overall, lens rubbing with MPS before soaking did not reduce the protein content on the lenses compared to nonrubbed lenses (p=0.89). PMID:20098668

Divergent selection for residual feed intake affects the transcriptomic and proteomic profiles of pig skeletal muscle.

PubMed

Vincent, A; Louveau, I; Gondret, F; Tréfeu, C; Gilbert, H; Lefaucheur, L

2015-06-01

Improving feed efficiency is a relevant strategy to reduce feed cost and environmental waste in livestock production. Selection experiments on residual feed intake (RFI), a measure of feed efficiency, previously indicated that low RFI was associated with lower feed intake, similar growth rate, and greater lean meat content compared with high RFI. To gain insights into the molecular mechanisms underlying these differences, 24 Large White females from 2 lines divergently selected for RFI were examined. Pigs from a low-RFI ("efficient") and high-RFI ("inefficient") line were individually fed ad libitum from 67 d of age (27 kg BW) to slaughter at 115 kg BW (n = 8 per group). Additional pigs of the high-RFI line were feed restricted to the daily feed intake of the ad libitum low-RFI pigs (n = 8) to investigate the impact of selection independently of feed intake. Global gene and protein expression profiles were assessed in the LM collected at slaughter. The analyses involved a porcine commercial microarray and 2-dimensional gel electrophoresis. About 1,000 probes were differentially expressed (P < 0.01) between RFI lines. Only 10% of those probes were also affected by feed restriction. Gene functional classification indicated a greater expression of genes involved in protein synthesis and a lower expression of genes associated with mitochondrial energy metabolism in the low-RFI pigs compared with the high-RFI pigs. At the protein level, 11 unique identified proteins exhibited a differential abundance (P < 0.05) between RFI lines. Differentially expressed proteins were generally not significantly affected by feed restriction. Mitochondrial oxidative proteins such as aconitase hydratase, ATP synthase subunit α, and creatine kinase S-type had a lower abundance in the low-RFI pigs, whereas fructose-biphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase, 2 proteins involved in glycolysis, had a greater abundance in those pigs compared with high-RFI pigs. Antioxidant proteins such as superoxide dismutase and glutathione peroxidase 3 at the mRNA level and peroxiredoxin-6 at the protein level were also less expressed in LM of the most efficient pigs, likely related to lower oxidative molecule production. Collectively, both the transcriptomic and proteomic approaches revealed a lower oxidative metabolism in muscle of the low-RFI pigs and all these modifications were largely independent of differences in feed intake.

Angle-resolved diffraction grating biosensor based on porous silicon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lv, Changwu; Li, Peng; Jia, Zhenhong, E-mail: jzhh@xju.edu.cn

2016-03-07

In this study, an optical biosensor based on a porous silicon composite structure was fabricated using a simple method. This structure consists of a thin, porous silicon surface diffraction grating and a one-dimensional porous silicon photonic crystal. An angle-resolved diffraction efficiency spectrum was obtained by measuring the diffraction efficiency at a range of incident angles. The angle-resolved diffraction efficiency of the 2nd and 3rd orders was studied experimentally and theoretically. The device was sensitive to the change of refractive index in the presence of a biomolecule indicated by the shift of the diffraction efficiency spectrum. The sensitivity of this sensormore » was investigated through use of an 8 base pair antifreeze protein DNA hybridization. The shifts of the angle-resolved diffraction efficiency spectrum showed a relationship with the change of the refractive index, and the detection limit of the biosensor reached 41.7 nM. This optical device is highly sensitive, inexpensive, and simple to fabricate. Using shifts in diffraction efficiency spectrum to detect biological molecules has not yet been explored, so this study establishes a foundation for future work.« less

Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production.

PubMed

Noftsger, S; St-Pierre, N R

2003-03-01

Metabolizable protein (MP) supply and amino acid balance were manipulated through selection of highly digestible rumen-undegradable protein (RUP) sources and methionine (Met) supplementation. Effects on production efficiency and N utilization of lactating dairy cows were determined. Thirty-two multiparous (647 kg) and 28 primiparous (550 kg) Holstein cows were assigned during the fourth week of lactation to one of four dietary treatments. Treatments were 1) 18.3% crude protein (CP) with low estimated intestinal digestibility of RUP (HiCP-LoDRUP), 2) 18.3% CP with high digestibility RUP (HiCP-HiDRUP), 3) 16.9% CP with high digestibility RUP (LoCP-HiDRUP), and 4) 17.0% CP with high digestibility RUP and supplemental Met (LoCP-HiDRUP + Met). Diets were balanced to have equal concentrations of net energy for lactation (NE(L)), acid detergent fiber (ADF), neutral detergent fiber (NDF), and ash. Milk yields (40.8, 46.2, 42.9, 46.6 kg/d), protein percentages (2.95, 2.98, 2.99, 3.09%), and fat percentages (3.42, 3.64, 3.66, 3.73%) are reported here for HiCP-LoDRUP, HiCP-HiDRUP, LoCP-HiDRUP, and LoCP-HiDRUP + Met, respectively. Milk urea N and BUN decreased when feeding a lower CP diet. Efficiency of use of N for milk protein production was higher when feeding higher digestibility RUP, especially with the LoCP-HiDRUP + Met diet. A digestibility study followed the production trial, with six cows per treatment group continuing on the same treatment for an additional week. The experimental periods were 5 d long, with 1 d of adjustment and 4 d of total collection of urine and feces. Dry matter intake, milk production, milk protein production, and N digestibility were not significantly different among treatments during the collection trial, whereas N intake and N absorbed increased with the higher CP diets. The quantity of N in feces did not change with diet, but quantity of N in urine decreased in the low CP diets. Milk N as a percentage of intake N and milk N as a percentage of N absorbed showed a trend toward increasing as CP concentration in the diet decreased. The supplementation of Met did not improve the efficiency of N utilization during the digestibility study, in contrast to what was estimated during the production trial. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows during the production trial, indicating that postruminal digestibility of RUP and amino acid balance can be more important than total RUP supplementation.

Protein corona: Opportunities and challenges

PubMed Central

Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M.; Mahmoudi, Morteza

2017-01-01

In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. PMID:26783938

Fluorescence Quantum Yield Measurements of Fluorescent Proteins: A Laboratory Experiment for a Biochemistry or Molecular Biophysics Laboratory Course

ERIC Educational Resources Information Center

Wall, Kathryn P.; Dillon, Rebecca; Knowles, Michelle K.

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts…

Biomarker Discovery and Verification of Esophageal Squamous Cell Carcinoma Using Integration of SWATH/MRM.

PubMed

Hou, Guixue; Lou, Xiaomin; Sun, Yulin; Xu, Shaohang; Zi, Jin; Wang, Quanhui; Zhou, Baojin; Han, Bo; Wu, Lin; Zhao, Xiaohang; Lin, Liang; Liu, Siqi

2015-09-04

We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

PubMed

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor.

PubMed

Quitterer, Ursula; Pohl, Armin; Langer, Andreas; Koller, Samuel; Abdalla, Said

2011-06-10

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer. Copyright © 2011 Elsevier Inc. All rights reserved.

Stability of Proteins in Carbohydrates and Other Additives during Freezing: The Human Growth Hormone as a Case Study.

PubMed

Arsiccio, Andrea; Pisano, Roberto

2017-09-21

Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.

Installing hydrolytic activity into a completely de novo protein framework

NASA Astrophysics Data System (ADS)

Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

2016-09-01

The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

PubMed

Golovko, A O; Koroleva, O N; Drutsa, V L

2017-12-01

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His) 6 -tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

PubMed

Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

2015-12-30

Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

Enrichment and separation techniques for large-scale proteomics analysis of the protein post-translational modifications.

PubMed

Huang, Junfeng; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

2014-11-06

Comprehensive analysis of the post-translational modifications (PTMs) on proteins at proteome level is crucial to elucidate the regulatory mechanisms of various biological processes. In the past decades, thanks to the development of specific PTM enrichment techniques and efficient multidimensional liquid chromatography (LC) separation strategy, the identification of protein PTMs have made tremendous progress. A huge number of modification sites for some major protein PTMs have been identified by proteomics analysis. In this review, we first introduced the recent progresses of PTM enrichment methods for the analysis of several major PTMs including phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and oxidation/reduction status. We then briefly summarized the challenges for PTM enrichment. Finally, we introduced the fractionation and separation techniques for efficient separation of PTM peptides in large-scale PTM analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel system for water soluble protein encapsulation with high efficiency: "micelles enhanced" polyelectrolyte capsules.

PubMed

Li, Xiaodong; Li, Xiaohui; Zhang, Jianxiang; Zhao, Shifang; Shen, Jiacong

2008-06-01

Novel "micelles enhanced" polyelectrolyte (PE) capsules based on functional templates of hybrid calcium carbonate were fabricated. Evidences suggested that the structure of capsule wall was different from that of conventional PE capsules, and the wall permeability of these PE capsules changed significantly. Lysozyme, a positively charged protein in neutral solution, was studied as a model protein to be encapsulated into the "micelles enhanced" PE capsules. Confocal laser scanning microscope was used to observe the entrapping process in real time, while UV-Vis spectroscope and scanning force microscope measurements suggested the high efficiency of encapsulation. In addition, the fluorescence recovery after photobleaching technique was employed to determine the existence form of deposited molecules. Further studies showed even negatively charged water-soluble peptides or proteins can be encapsulated into these hybrid capsules by modulating the pH value in bulk solution under its isoelectronic point as well. Copyright 2007 Wiley Periodicals, Inc.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

PubMed Central

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-01-01

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

PubMed

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-07-23

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.

Nanocapillaries for Open Tubular Chromatographic Separations of Proteins in Femtoliter to Picoliter Samples

PubMed Central

Wang, Xiayan; Cheng, Chang; Wang, Shili; Zhao, Meiping; Dasgupta, Purnendu K.; Liu, Shaorong

2009-01-01

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 106 theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 μm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto to picoliter (fL-pL) volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typ. 200 psi) as the pump and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit μL volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, α-lactalbumin, insulin, and α -2-macroglobulin. PMID:19663450

On plate graphite supported sample processing for simultaneous lipid and protein identification by matrix assisted laser desorption ionization mass spectrometry.

PubMed

Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Sabbatini, Luigia; Palmisano, Francesco

2015-05-01

The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15 min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Optical Lock-In Detection of FRET Using Synthetic and Genetically Encoded Optical Switches

PubMed Central

Mao, Shu; Benninger, Richard K. P.; Yan, Yuling; Petchprayoon, Chutima; Jackson, David; Easley, Christopher J.; Piston, David W.; Marriott, Gerard

2008-01-01

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins. PMID:18281383

Stability and the Evolvability of Function in a Model Protein

PubMed Central

Bloom, Jesse D.; Wilke, Claus O.; Arnold, Frances H.; Adami, Christoph

2004-01-01

Functional proteins must fold with some minimal stability to a structure that can perform a biochemical task. Here we use a simple model to investigate the relationship between the stability requirement and the capacity of a protein to evolve the function of binding to a ligand. Although our model contains no built-in tradeoff between stability and function, proteins evolved function more efficiently when the stability requirement was relaxed. Proteins with both high stability and high function evolved more efficiently when the stability requirement was gradually increased than when there was constant selection for high stability. These results show that in our model, the evolution of function is enhanced by allowing proteins to explore sequences corresponding to marginally stable structures, and that it is easier to improve stability while maintaining high function than to improve function while maintaining high stability. Our model also demonstrates that even in the absence of a fundamental biophysical tradeoff between stability and function, the speed with which function can evolve is limited by the stability requirement imposed on the protein. PMID:15111394

Genome engineering for improved recombinant protein expression in Escherichia coli.

PubMed

Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

2014-12-19

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

The Taming of the Cell Penetrating Domain of the HIV Tat: Myths and Realities

PubMed Central

Chauhan, Ashok; Tikoo, Akshay; Kapur, Arvinder K.; Singh, Mahavir

2007-01-01

Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences. PMID:17196289

Chapter 16: Soy Proteins as Wood Adhesives

Treesearch

Charles R. Frihart; Christopher G. Hunt; Michael J. Birkeland

2014-01-01

Protein adhesives allowed the development of bonded wood products such as plywood and glulam in the early 20th century. Petrochemical-based adhesives replaced proteins in most wood bonding applications because of lower cost, improved production efficiencies, and enhanced durability. However, several technological and environmental factors have led to a resurgence of...

Growth of Atlantic salmon, Salmo salar fed diets containing barley protein concentrate

USDA-ARS?s Scientific Manuscript database

Atlantic salmon (Salmo salar) is an important cultured carnivorous species that in the past has not tolerated high levels of most plant protein feed ingredients in the diet. In order to increase efficiency, sustainability and production to meet global demand, new sources of protein must be incorpo...

Efficiency of dairy farms participating and not participating in veterinary herd health management programs.

PubMed

Derks, Marjolein; Hogeveen, Henk; Kooistra, Sake R; van Werven, Tine; Tauer, Loren W

2014-12-01

This paper compares farm efficiencies between dairies who were participating in a veterinary herd health management (VHHM) program with dairies not participating in such a program, to determine whether participation has an association with farm efficiency. In 2011, 572 dairy farmers received a questionnaire concerning the participation and execution of a VHHM program on their farms. Data from the questionnaire were combined with farm accountancy data from 2008 through 2012 from farms that used calendar year accounting periods, and were analyzed using Stochastic Frontier Analysis (SFA). Two separate models were specified: model 1 was the basic stochastic frontier model (output: total revenue; input: feed costs, land costs, cattle costs, non-operational costs), without explanatory variables embedded into the efficiency component of the error term. Model 2 was an expansion of model 1 which included explanatory variables (number of FTE; total kg milk delivered; price of concentrate; milk per hectare; cows per FTE; nutritional yield per hectare) inserted into the efficiency component of the joint error term. Both models were estimated with the financial parameters expressed per 100 kg fat and protein corrected milk and per cow. Land costs, cattle costs, feed costs and non-operational costs were statistically significant and positive in all models (P<0.01). Frequency distributions of the efficiency scores for the VHHM dairies and the non-VHHM dairies were plotted in a kernel density plot, and differences were tested using the Kolmogorov-Smirnov two-sample test. VHHM dairies had higher total revenue per cow, but not per 100 kg milk. For all SFA models, the difference in distribution was not statistically different between VHHM dairies and non-VHHM dairies (P values 0.94, 0.35, 0.95 and 0.89 for the basic and complete model per 100 kg fat and protein corrected milk and per cow respectively). Therefore we conclude that with our data farm participation in VHHM is not related to overall farm efficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates human immunodeficiency virus type 1 infection

PubMed Central

2012-01-01

Background Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified. Results In this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNALys3 into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNALys3 into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55gag and p160gag-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood mononuclear cells (PBMCs). Conclusions These findings indicate that GAPDH negatively regulates HIV-1 infection and provide insights into a novel function of GAPDH in the HIV-1 life cycle and a new host defense mechanism against HIV-1 infection. PMID:23237566

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

PubMed

Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

2017-03-02

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Can direct conversion of used nitrogen to new feed and protein help feed the world?

PubMed

Matassa, Silvio; Batstone, Damien J; Hülsen, Tim; Schnoor, Jerald; Verstraete, Willy

2015-05-05

The increase in the world population, vulnerability of conventional crop production to climate change, and population shifts to megacities justify a re-examination of current methods of converting reactive nitrogen to dinitrogen gas in sewage and waste treatment plants. Indeed, by up-grading treatment plants to factories in which the incoming materials are first deconstructed to units such as ammonia, carbon dioxide and clean minerals, one can implement a highly intensive and efficient microbial resynthesis process in which the used nitrogen is harvested as microbial protein (at efficiencies close to 100%). This can be used for animal feed and food purposes. The technology for recovery of reactive nitrogen as microbial protein is available but a change of mindset needs to be achieved to make such recovery acceptable.

[AFM fishing of proteins under impulse electric field].

PubMed

Ivanov, Yu D; Pleshakova, T O; Malsagova, K A; Kaysheva, A L; Kopylov, A T; Izotov, A A; Tatur, V Yu; Vesnin, S G; Ivanova, N D; Ziborov, V S; Archakov, A I

2016-05-01

A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

Verification of 2A peptide cleavage.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, P.; Whitwam, R.E.; Tien, Ming

1996-03-01

A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

Efficient Simulation of Explicitly Solvated Proteins in the Well-Tempered Ensemble.

PubMed

Deighan, Michael; Bonomi, Massimiliano; Pfaendtner, Jim

2012-07-10

Herein, we report significant reduction in the cost of combined parallel tempering and metadynamics simulations (PTMetaD). The efficiency boost is achieved using the recently proposed well-tempered ensemble (WTE) algorithm. We studied the convergence of PTMetaD-WTE conformational sampling and free energy reconstruction of an explicitly solvated 20-residue tryptophan-cage protein (trp-cage). A set of PTMetaD-WTE simulations was compared to a corresponding standard PTMetaD simulation. The properties of PTMetaD-WTE and the convergence of the calculations were compared. The roles of the number of replicas, total simulation time, and adjustable WTE parameter γ were studied.

Multi-state lasing in self-assembled ring-shaped green fluorescent protein microcavities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dietrich, Christof P., E-mail: cpd3@st-andrews.ac.uk; Höfling, Sven; Gather, Malte C., E-mail: mcg6@st-andrews.ac.uk

2014-12-08

We demonstrate highly efficient lasing from multiple photonic states in microcavities filled with self-assembled rings of recombinant enhanced green fluorescent protein (eGFP) in its solid state form. The lasing regime is achieved at very low excitation energies of 13 nJ and occurs from cavity modes dispersed in both energy and momentum. We attribute the momentum distribution to very efficient scattering of incident light at the surface of the eGFP rings. The distribution of lasing states in energy is induced by the large spectral width of the gain spectrum of recombinant eGFP (FWHM ≅ 25 nm)

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

PubMed

Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross

2017-08-25

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

NASA Astrophysics Data System (ADS)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

PubMed Central

Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

2007-01-01

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

PubMed

Lee, C K; Knipe, D M

1985-06-01

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments.

PubMed

Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

2003-11-01

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

Binding free energy predictions of farnesoid X receptor (FXR) agonists using a linear interaction energy (LIE) approach with reliability estimation: application to the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Rifai, Eko Aditya; van Dijk, Marc; Vermeulen, Nico P. E.; Geerke, Daan P.

2018-01-01

Computational protein binding affinity prediction can play an important role in drug research but performing efficient and accurate binding free energy calculations is still challenging. In the context of phase 2 of the Drug Design Data Resource (D3R) Grand Challenge 2 we used our automated eTOX ALLIES approach to apply the (iterative) linear interaction energy (LIE) method and we evaluated its performance in predicting binding affinities for farnesoid X receptor (FXR) agonists. Efficiency was obtained by our pre-calibrated LIE models and molecular dynamics (MD) simulations at the nanosecond scale, while predictive accuracy was obtained for a small subset of compounds. Using our recently introduced reliability estimation metrics, we could classify predictions with higher confidence by featuring an applicability domain (AD) analysis in combination with protein-ligand interaction profiling. The outcomes of and agreement between our AD and interaction-profile analyses to distinguish and rationalize the performance of our predictions highlighted the relevance of sufficiently exploring protein-ligand interactions during training and it demonstrated the possibility to quantitatively and efficiently evaluate if this is achieved by using simulation data only.

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed Central

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-01-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery. Images PMID:8599930

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-02-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

PubMed

Tran, Lee; Hanavan, Paul D; Campbell, Latoya E; De Filippis, Elena; Lake, Douglas F; Coletta, Dawn K; Roust, Lori R; Mandarino, Lawrence J; Carroll, Chad C; Katsanos, Christos S

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

PubMed Central

Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

2007-01-01

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes. PMID:17381426

Disrupting the PCSK9/LDLR protein-protein interaction by an imidazole-based minimalist peptidomimetic.

PubMed

Stucchi, Mattia; Grazioso, Giovanni; Lammi, Carmen; Manara, Silvia; Zanoni, Chiara; Arnoldi, Anna; Lesma, Giordano; Silvani, Alessandra

2016-10-18

Herein we report on the multicomponent synthesis of a novel imidazole-based compound, able to act efficiently as a minimalist β-strand mimic. Biological evaluation proved its ability to impair the LDLR-PCSK9 protein-protein interaction, disclosing it as the first small molecule exerting a PCSK9-mediated hypocholesterolemic effect.

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

PubMed Central

Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

2015-01-01

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

Effects of two commercially available feline diets on glucose and insulin concentrations, insulin sensitivity and energetic efficiency of weight gain.

PubMed

Coradini, M; Rand, J S; Morton, J M; Rawlings, J M

2011-10-01

A low-carbohydrate, high-protein (LCHP) diet is often recommended for the prevention and management of diabetes in cats; however, the effect of macronutrient composition on insulin sensitivity and energetic efficiency for weight gain is not known. The present study compared the effect in adult cats (n 32) of feeding a LCHP (23 and 47 % metabolisable energy (ME)) and a high-carbohydrate, low-protein (HCLP) diet (51 and 21 % ME) on fasting and postprandial glucose and insulin concentrations, and on insulin sensitivity. Tests were done in the 4th week of maintenance feeding and after 8 weeks of ad libitum feeding, when weight gain and energetic efficiency of each diet were also measured. When fed at maintenance energy, the HCLP diet resulted in higher postprandial glucose and insulin concentrations. When fed ad libitum, the LCHP diet resulted in greater weight gain (P < 0.01), and was associated with higher energetic efficiency. Overweight cats eating the LCHP diet had similar postprandial glucose concentrations to lean cats eating the HCLP diet. Insulin sensitivity was not different between the diets when cats were lean or overweight, but glucose effectiveness was higher after weight gain in cats fed the HCLP diet. According to the present results, LCHP diets fed at maintenance requirements might benefit cats with multiple risk factors for developing diabetes. However, ad libitum feeding of LCHP diets is not recommended as they have higher energetic efficiency and result in greater weight gain.

Fetal and neonatal programming of postnatal growth and feed efficiency in swine.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Wang, Xiaolong; Li, Ju; Wang, Binggen; Wu, Guoyao

2017-01-01

Maternal undernutrition or overnutrition during pregnancy alters organ structure, impairs prenatal and neonatal growth and development, and reduces feed efficiency for lean tissue gains in pigs. These adverse effects may be carried over to the next generation or beyond. This phenomenon of the transgenerational impacts is known as fetal programming, which is mediated by stable and heritable alterations of gene expression through covalent modifications of DNA and histones without changes in DNA sequences (namely, epigenetics). The mechanisms responsible for the epigenetic regulation of protein expression and functions include chromatin remodeling; DNA methylation (occurring at the 5´-position of cytosine residues within CpG dinucleotides); and histone modifications (acetylation, methylation, phosphorylation, and ubiquitination). Like maternal malnutrition, undernutrition during the neonatal period also reduces growth performance and feed efficiency (weight gain:feed intake; also known as weight-gain efficiency) in postweaning pigs by 5-10%, thereby increasing the days necessary to reach the market body-weight. Supplementing functional amino acids (e.g., arginine and glutamine) and vitamins (e.g., folate) play a key role in activating the mammalian target of rapamycin signaling and regulating the provision of methyl donors for DNA and protein methylation. Therefore, these nutrients are beneficial for the dietary treatment of metabolic disorders in offspring with intrauterine growth restriction or neonatal malnutrition. The mechanism-based strategies hold great promise for the improvement of the efficiency of pork production and the sustainability of the global swine industry.

Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

PubMed Central

Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

2016-01-01

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

Protein export through the bacterial flagellar type III export pathway.

PubMed

Minamino, Tohru

2014-08-01

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.

Unraveling the Fecal Microbiota and Metagenomic Functional Capacity Associated with Feed Efficiency in Pigs

PubMed Central

Yang, Hui; Huang, Xiaochang; Fang, Shaoming; He, Maozhang; Zhao, Yuanzhang; Wu, Zhenfang; Yang, Ming; Zhang, Zhiyan; Chen, Congying; Huang, Lusheng

2017-01-01

Gut microbiota plays fundamental roles in energy harvest, nutrient digestion, and intestinal health, especially in processing indigestible components of polysaccharides in diet. Unraveling the microbial taxa and functional capacity of gut microbiome associated with feed efficiency can provide important knowledge to improve pig feed efficiency in swine industry. In the current research, we studied the association of fecal microbiota with feed efficiency in 280 commercial Duroc pigs. All experimental pigs could be clustered into two enterotype-like groups. Different enterotypes showed the tendency of association with the feed efficiency (P = 0.07). We further identified 31 operational taxonomic units (OTUs) showing the potential associations with porcine feed efficiency. These OTUs were mainly annotated to the bacteria related to the metabolisms of dietary polysaccharides. Although we did not identify the RFI-associated bacterial species at FDR < 0.05 level, metagenomic sequencing analysis did find the distinct function capacities of gut microbiome between the high and low RFI pigs (FDR < 0.05). The KEGG orthologies related to nitrogen metabolism, amino acid metabolism, and transport system, and eight KEGG pathways including glycine, serine, and threonine metabolism were positively associated with porcine feed efficiency. We inferred that gut microbiota might improve porcine feed efficiency through promoting intestinal health by the SCFAs produced by fermenting dietary polysaccharides and improving the utilization of dietary protein. The present results provided important basic knowledge for improving porcine feed efficiency through modulating gut microbiome. PMID:28861066

Qualitative and quantitative evaluation of derivatization reagents for different types of protein-bound carbonyl groups.

PubMed

Bollineni, Ravi Chand; Fedorova, Maria; Hoffmann, Ralf

2013-09-07

Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.

Protein-functionalized magnetic iron oxide nanoparticles: time efficient potential-water treatment

NASA Astrophysics Data System (ADS)

Okoli, Chuka; Boutonnet, Magali; Järås, Sven; Rajarao-Kuttuva, Gunaratna

2012-10-01

Recent advances in nanoscience suggest that the existing issues involving water quality could be resolved or greatly improved using nanomaterials, especially magnetic iron oxide nanoparticles. Magnetic nanoparticles have been synthesized for the development and use, in association with natural coagulant protein for water treatment. The nanoparticles size, morphology, structure, and magnetic properties were characterized by transmission electron microscope, X-ray diffraction, and superconducting quantum interference device magnetometry. Purified Moringa oleifera protein was attached onto microemulsions-prepared magnetic iron oxide nanoparticles (ME-MION) to form stable protein-functionalized magnetic nanoparticles (PMO+ME-MION). The turbidity removal efficiency in both synthetic and surface water samples were investigated and compared with the commonly used synthetic coagulant (alum) as well as PMO. More than 90 % turbidity could be removed from the surface waters within 12 min by magnetic separation of PMO+ME-MION; whereas gravimetrically, 70 % removal in high and low turbid waters can be achieved within 60 min. In contrast, alum requires 180 min to reduce the turbidity of low turbid water sample. These data support the advantage of separation with external magnetic field (magnetophoresis) over gravitational force. Time kinetics studies show a significant enhancement in ME-MION efficiency after binding with PMO implying the availability of large surface of the ME-MION. The coagulated particles (impurities) can be removed from PMO+ME-MION by washing with mild detergent or cleaning solution. To our knowledge, this is the first report on surface water turbidity removal using protein-functionalized magnetic nanoparticle.

3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

PubMed

Nagae, Masamichi; Hirata, Tetsuya; Morita-Matsumoto, Kana; Theiler, Romina; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

2016-10-09

The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (K d = ~10 -4 M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-adaption of tRNA gene copy number and amino acid usage influences translation rates in three life domains.

PubMed

Du, Meng-Ze; Wei, Wen; Qin, Lei; Liu, Shuo; Zhang, An-Ying; Zhang, Yong; Zhou, Hong; Guo, Feng-Biao

2017-12-01

Although more and more entangled participants of translation process were realized, how they cooperate and co-determine the final translation efficiency still lacks details. Here, we reasoned that the basic translation components, tRNAs and amino acids should be consistent to maximize the efficiency and minimize the cost. We firstly revealed that 310 out of 410 investigated genomes of three domains had significant co-adaptions between the tRNA gene copy numbers and amino acid compositions, indicating that maximum efficiency constitutes ubiquitous selection pressure on protein translation. Furthermore, fast-growing and larger bacteria are found to have significantly better co-adaption and confirmed the effect of this pressure. Within organism, highly expressed proteins and those connected to acute responses have higher co-adaption intensity. Thus, the better co-adaption probably speeds up the growing of cells through accelerating the translation of special proteins. Experimentally, manipulating the tRNA gene copy number to optimize co-adaption between enhanced green fluorescent protein (EGFP) and tRNA gene set of Escherichia coli indeed lifted the translation rate (speed). Finally, as a newly confirmed translation rate regulating mechanism, the co-adaption reflecting translation rate not only deepens our understanding on translation process but also provides an easy and practicable method to improve protein translation rates and productivity. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

PubMed

Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

2015-11-19

Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

NASA Astrophysics Data System (ADS)

El-Niely, Hania F. G.

2007-06-01

The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas ( Pisum satinum L), cowpeas ( Vigna unguiculata L.Walp), lentils ( Lens culinaris Med), kidneybeans ( Phaseolus vulgaris L), and chickpeas ( Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly ( p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

PubMed

Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

2010-07-01

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters.

PubMed

Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang

2014-10-01

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml(-1)) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.

Berkeley Screen: a set of 96 solutions for general macromolecular crystallization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Jose H.; McAndrew, Ryan P.; Tomaleri, Giovani P.

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography programmore » at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens. The Berkeley Screen provides an efficient set of solutions for general macromolecular crystallization trials.« less

Displacers improve the selectivity of phosphopeptide enrichment by metal oxide affinity chromatography.

PubMed

Herrera, Yesenia; Contreras, Sandra; Hernández, Magdalena; Álvarez, Laura; Mora, Yolanda; Encarnación-Guevara, Sergio

A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites. We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO 2 )-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO 2 and phosphorylated peptides. The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument. This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Berkeley Screen: a set of 96 solutions for general macromolecular crystallization

DOE PAGES

Pereira, Jose H.; McAndrew, Ryan P.; Tomaleri, Giovani P.; ...

2017-09-05

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography programmore » at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens. The Berkeley Screen provides an efficient set of solutions for general macromolecular crystallization trials.« less

Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

PubMed

Ohba, M

1997-06-09

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms☆

PubMed Central

Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

2016-01-01

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins. Guest Editors: J.C. Gumbart and Sergei Noskov. PMID:26766517

Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo.

PubMed

Shi, Ming; Zhang, Ling; Gu, Hong-Tao; Jiang, Feng-Qin; Qian, Lu; Yu, Ming; Chen, Guo-Jiang; Luo, Qun; Shen, Bei-Fen; Guo, Ning

2007-10-01

To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo. Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models. The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo. The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

The dual role of paramagnetic particles for integrated lysis and measurement in a rapid immunoassay for intracellular proteins.

PubMed

Sharif, Elham; Kiely, Janice; Wraith, Patrick; Luxton, Richard

2013-05-01

A novel, integrated lysis and immunoassay methodology and system for intracellular protein measurement are described. The method uses paramagnetic particles both as a lysis agent and assay label resulting in a rapid test requiring minimal operator intervention, the test being homogeneous and completed in less than 10 min. A design study highlights the critical features of the magnetic detection system used to quantify the paramagnetic particles and a novel frequency-locked loop-based magnetometer is presented. A study of paramagnetic particle enhanced lysis demonstrates that the technique is more than twice as efficient at releasing intracellular protein as ultrasonic lysis alone. Results are presented for measurements of intracellular prostate specific antigen in an LNCAP cell line. This model was selected to demonstrate the rapidity and efficiency of intracellular protein quantification. It was shown that, on average, LNCAP cells contained 0.43 fg of prostate specific antigen. This system promises an attractive solution for applications that require a rapid determination of intracellular proteins.

Kernel based machine learning algorithm for the efficient prediction of type III polyketide synthase family of proteins.

PubMed

Mallika, V; Sivakumar, K C; Jaichand, S; Soniya, E V

2010-07-13

Type III Polyketide synthases (PKS) are family of proteins considered to have significant roles in the biosynthesis of various polyketides in plants, fungi and bacteria. As these proteins shows positive effects to human health, more researches are going on regarding this particular protein. Developing a tool to identify the probability of sequence being a type III polyketide synthase will minimize the time consumption and manpower efforts. In this approach, we have designed and implemented PKSIIIpred, a high performance prediction server for type III PKS where the classifier is Support Vector Machines (SVMs). Based on the limited training dataset, the tool efficiently predicts the type III PKS superfamily of proteins with high sensitivity and specificity. The PKSIIIpred is available at http://type3pks.in/prediction/. We expect that this tool may serve as a useful resource for type III PKS researchers. Currently work is being progressed for further betterment of prediction accuracy by including more sequence features in the training dataset.

Protein Sequence Classification with Improved Extreme Learning Machine Algorithms

PubMed Central

2014-01-01

Precisely classifying a protein sequence from a large biological protein sequences database plays an important role for developing competitive pharmacological products. Comparing the unseen sequence with all the identified protein sequences and returning the category index with the highest similarity scored protein, conventional methods are usually time-consuming. Therefore, it is urgent and necessary to build an efficient protein sequence classification system. In this paper, we study the performance of protein sequence classification using SLFNs. The recent efficient extreme learning machine (ELM) and its invariants are utilized as the training algorithms. The optimal pruned ELM is first employed for protein sequence classification in this paper. To further enhance the performance, the ensemble based SLFNs structure is constructed where multiple SLFNs with the same number of hidden nodes and the same activation function are used as ensembles. For each ensemble, the same training algorithm is adopted. The final category index is derived using the majority voting method. Two approaches, namely, the basic ELM and the OP-ELM, are adopted for the ensemble based SLFNs. The performance is analyzed and compared with several existing methods using datasets obtained from the Protein Information Resource center. The experimental results show the priority of the proposed algorithms. PMID:24795876

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

PubMed

Jean, M; Smaoui, F; Lavertu, M; Méthot, S; Bouhdoud, L; Buschmann, M D; Merzouki, A

2009-09-01

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency.

PubMed

Jensen, Kristopher Torp; Fløe, Lasse; Petersen, Trine Skov; Huang, Jinrong; Xu, Fengping; Bolund, Lars; Luo, Yonglun; Lin, Lin

2017-07-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design. © 2017 Federation of European Biochemical Societies.

Web application for automatic prediction of gene translation elongation efficiency.

PubMed

Sokolov, Vladimir; Zuraev, Bulat; Lashin, Sergei; Matushkin, Yury

2015-09-03

Expression efficiency is one of the major characteristics describing genes in various modern investigations. Expression efficiency of genes is regulated at various stages: transcription, translation, posttranslational protein modification and others. In this study, a special EloE (Elongation Efficiency) web application is described. The EloE sorts the organism's genes in a descend order on their theoretical rate of the elongation stage of translation based on the analysis of their nucleotide sequences. Obtained theoretical data have a significant correlation with available experimental data of gene expression in various organisms. In addition, the program identifies preferential codons in organism's genes and defines distribution of potential secondary structures energy in 5´ and 3´ regions of mRNA. The EloE can be useful in preliminary estimation of translation elongation efficiency for genes for which experimental data are not available yet. Some results can be used, for instance, in other programs modeling artificial genetic structures in genetically engineered experiments.

Developing an in silico model of the modulation of base excision repair using methoxyamine for more targeted cancer therapeutics.

PubMed

Gurkan-Cavusoglu, Evren; Avadhani, Sriya; Liu, Lili; Kinsella, Timothy J; Loparo, Kenneth A

2013-04-01

Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763