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.

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

PubMed

Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

2016-04-19

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns

PubMed Central

Shinomiya, Kazufusa; Umezawa, Motoki; Seki, Manami; Nitta, Jun; Zaima, Kazumasa; Harikai, Naoki; Ito, Yoichiro

2016-01-01

1) Background Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time. 2) Methods The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge. 3) Results Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention. 4) Conclusion Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule. PMID:27891507

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.

Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

NASA Astrophysics Data System (ADS)

Ouyang, Ruizhuo; Lei, Jianping; Ju, Huangxian

2010-05-01

This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 × 1018 g - 1, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

PubMed Central

Park, Sung-Soo; Maudsley, Stuart

2010-01-01

Global profiling of phosphoproteomes has proven a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically-relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography (TOC) can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH-gradient elution technique for enhanced phosphopeptide identification in conjunction with TOC. Using this process, we have demonstrated its superiority to the traditional ‘one-pot’ strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH-gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pH 9.2 and 9.4. High-resolution separation of multiply-phosphorylated peptides was primarily achieved using elution ranges > pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values. PMID:20946866

Design and engineering of water-soluble light-harvesting protein maquettes

DOE PAGES

Kodali, Goutham; Mancini, Joshua A.; Solomon, Lee A.; ...

2017-01-01

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistrymore » of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. In conclusion, this partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.« less

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.

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistrymore » of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. In conclusion, this partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.« less

Acoustic technology for high-performance disruption and extraction of plant proteins.

PubMed

Toorchi, Mahmoud; Nouri, Mohammad-Zaman; Tsumura, Makoto; Komatsu, Setsuko

2008-07-01

Acoustic technology shows the capability of protein pellet homogenization from different tissue samples of soybean and rice in a manner comparable to the ordinary mortar/pestle method and far better than the vortex/ultrasonic method with respect to the resolution of the protein pattern through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). With acoustic technology, noncontact tissue disruption and protein pellet homogenization can be carried out in a computer-controlled manner, which ultimately increases the efficiency of the process for a large number of samples. A lysis buffer termed the T-buffer containing TBP, thiourea, and CHAPS yields an excellent result for the 2D-PAGE separation of soybean plasma membrane proteins followed by the 2D-PAGE separation of crude protein of soybean and rice tissues. For this technology, the T-buffer is preferred because protein quantification is possible by eliminating the interfering compound 2-mercaptoethanol and because of the high reproducibility of 2D-PAGE separation.

Applications of capillary electrophoresis in characterizing recombinant protein therapeutics.

PubMed

Zhao, Shuai Sherry; Chen, David D Y

2014-01-01

The use of recombinant protein for therapeutic applications has increased significantly in the last three decades. The heterogeneity of these proteins, often caused by the complex biosynthesis pathways and the subsequent PTMs, poses a challenge for drug characterization to ensure its safety, quality, integrity, and efficacy. CE, with its simple instrumentation, superior separation efficiency, small sample consumption, and short analysis time, is a well-suited analytical tool for therapeutic protein characterization. Different separation modes, including CIEF, SDS-CGE, CZE, and CE-MS, provide complementary information of the proteins. The CE applications for recombinant therapeutic proteins from the year 2000 to June 2013 are reviewed and technical concerns are discussed in this article. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein.

PubMed

Li, Na; Wang, Yuzhi; Xu, Kaijia; Huang, Yanhua; Wen, Qian; Ding, Xueqin

2016-05-15

Six kinds of new type of green betaine-based deep eutectic solvents (DESs) have been synthesized. Deep eutectic solvent aqueous two-phase systems (DES-ATPS) were established and successfully applied in the extraction of protein. Betaine-urea (Be-U) was selected as the suitable extractant. Single factor experiments were carried out to determine the optimum conditions of the extraction process, such as the salt concentration, the mass of DES, the separation time, the amount of protein, the temperature and the pH value. The extraction efficiency could achieve to 99.82% under the optimum conditions. Mixed sample and practical sample analysis were discussed. The back extraction experiment was implemented and the back extraction efficiency could reach to 32.66%. The precision experiment, repeatability experiment and stability experiment were investigated. UV-vis, FT-IR and circular dichroism (CD) spectra confirmed that the conformation of protein was not changed during the process of extraction. The mechanisms of extraction were researched by dynamic light scattering (DLS), the measurement of the conductivity and transmission electron microscopy (TEM). DES-protein aggregates and embraces phenomenon play considerable roles in the separation process. All of these results indicated that betaine-based DES-ATPS may provide a potential substitute new method for the separation of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Magnetic techniques for the isolation and purification of proteins and peptides

PubMed Central

Safarik, Ivo; Safarikova, Mirka

2004-01-01

Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. PMID:15566570

Selective binding and magnetic separation of His-tagged proteins using Fe3O4/PAM/NTA-Ni2+ Magnetic Nanoparticles

NASA Astrophysics Data System (ADS)

Guo, Huiling; Li, Mengyun; Tu, Shu; Sun, Honghao

2018-03-01

Fe3O4 nanoparticles coated with polyacrylamide (PAM) were synthesized. The magnetic core, with an average hydrodynamic size of 235.5 nm, allowed the magnetic nanoparticles (MNPs) rapid separation from solutions under an external magnetic field. NTA-Ni2+ was modified on the surface of Fe3O4/PAM MNPs to selectively trap his-tagged green fluorescent protein (GFP). The results showed that Fe3O4/PAM/NTA-Ni2+ MNPs exhibited remarkable capability of selective binding and separating his-tagged GFP. The adsorption efficiency was 93.37%.

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

Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids.

PubMed

Kortz, Linda; Helmschrodt, Christin; Ceglarek, Uta

2011-03-01

In the last decade various analytical strategies have been established to enhance separation speed and efficiency in high performance liquid chromatography applications. Chromatographic supports based on monolithic material, small porous particles, and porous layer beads have been developed and commercialized to improve throughput and separation efficiency. This paper provides an overview of current developments in fast chromatography combined with mass spectrometry for the analysis of metabolites and proteins in clinical applications. Advances and limitations of fast chromatography for the combination with mass spectrometry are discussed. Practical aspects of, recent developments in, and the present status of high-throughput analysis of human body fluids for therapeutic drug monitoring, toxicology, clinical metabolomics, and proteomics are presented.

Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes

PubMed Central

Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Durai, Prasannavenkatesh; Shah, Masaud; Choi, Sangdun

2016-01-01

The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming. PMID:26790000

Coupling Sodium Dodecyl Sulfate–Capillary Polyacrylamide Gel Electrophoresis with MALDI-TOF-MS via a PTFE Membrane

PubMed Central

Lu, Joann J.; Zhu, Zaifang; Wang, Wei; Liu, Shaorong

2011-01-01

Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS–capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI–TOF–MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes. PMID:21309548

Problems with multiple use of transfer buffer in protein electrophoretic transfer.

PubMed

Dorri, Yaser; Kurien, Biji T; Scofield, R Hal

2010-04-01

Two-dimensional gel electrophoresis (2DE) and SDS-PAGE are the two most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are usually transferred to membranes using a variety of methods, such as electrophoretic transfer, heat-mediated transfer, or nonelectrophoretic transfer, for specific protein detection and/or analysis. In a recent study, Pettegrew et al. claim to reuse transfer buffer containing methanol for at least five times for transferring proteins from SDS-PAGE to polyvinylidene difluoride. They add 150-200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is not accurate and useful for protein analysis, and it can cause background binding as well as inaccurate protein analysis.

Preparation and characterization of monodisperse large-porous silica microspheres as the matrix for protein separation.

PubMed

Xia, Hongjun; Wan, Guangping; Zhao, Junlong; Liu, Jiawei; Bai, Quan

2016-11-04

High performance liquid chromatography (HPLC) is a kind of efficient separation technology and has been used widely in many fields. Micro-sized porous silica microspheres as the most popular matrix have been used for fast separation and analysis in HPLC. In this paper, the monodisperse large-porous silica microspheres with controllable size and structure were successfully synthesized with polymer microspheres as the templates and characterized. First, the poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) microspheres (P GMA-EDMA ) were functionalized with tetraethylenepentamine (TEPA) to generate amino groups which act as a catalyst in hydrolysis of tetraethyl orthosilicate (TEOS) to form Si-containing low molecular weight species. Then the low molecular weight species diffused into the functionalized P GMA-EDMA microspheres by induction force of the amino groups to form polymer/silica hybrid microspheres. Finally, the organic polymer templates were removed by calcination, and the large-porous silica microspheres were obtained. The compositions, morphology, size distribution, specific surface area and pore size distribution of the porous silica microspheres were characterized by infrared analyzer, scanning-electron microscopy, dynamic laser scattering, the mercury intrusion method and thermal gravimetric analysis, respectively. The results show that the agglomeration of the hybrid microspheres can be overcome when the templates were functionalized with TEPA as amination reagent, and the yield of 95.7% of the monodisperse large-porous silica microspheres can be achieved with high concentration of polymer templates. The resulting large-porous silica microspheres were modified with octadecyltrichlorosilane (ODS) and the chromatographic evaluation was performed by separating the proteins and the digest of BSA. The baseline separation of seven kinds of protein standards was achieved, and the column delivered a better performance when separating BSA digests comparing with the commercial one currently available. The high column efficiency and good reproducibility present that the large-porous silica microspheres obtained can be used as a matrix for peptide and protein separation. Copyright © 2016 Elsevier B.V. All rights reserved.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

PubMed

Bright, G R; Spooner, B S

1983-06-01

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification.

PubMed

Zhang, Hu-Cheng; Yang, Jun; Yang, Guo-Wei; Wang, Xiao-Jie; Fan, Hai-Tao

2015-08-01

Recombinant Streptococcus Protein G (PG) is a cell wall protein, which, when combined with mammal immunoglobulin, is used in separating antibody technology. High-density fermentation technologies using an engineered recombinant PG-producing bacteria as well as PG separation and purification technologies have a direct impact on the availability and application of PG. Through primary and secondary seed cultivation, a recombinant E. coli strain was subjected to high-density fermentation with controlled feed supplement concentration under stimulation with isopropyl β-D-1-thiogalactopyranoside. The present study investigated the effect of factors including inoculum size, oxygen levels, pH and the cultivating method on the fermentation process, as well as the effect of the separation and purification technologies, including ultrasonication, nickel column affinity chromatography, Sephadex G-25 gel filtration chromatography and diethylaminoethanol-sepharose fast flow ion exchange chromatography on the yield and purity of PG. The efficiency of extraction was detected using SDS-PAGE. High-density fermentation yielded 80-150 g/l of bacteria and 1 g PG was obtained from one liter broth. The present study delivered a highly efficient novel method via which PG can be obtained at a high concentration and a purity >95%.

Electrospun nylon 6/zinc doped hydroxyapatite membrane for protein separation: Mechanism of fouling and blocking model.

PubMed

Esfahani, Hamid; Prabhakaran, Molamma P; Salahi, Esmaeil; Tayebifard, Ali; Rahimipour, Mohamad Reza; Keyanpour-Rad, Mansour; Ramakrishna, Seeram

2016-02-01

Development of composite nanofibrous membrane via electrospinning a polymer with ceramic nanoparticles (NPs) for application in protein separation systems is explored during this study. Positively charged zinc doped hydroxyapatite (xZH) NPs were prepared in three different compositions via chemical precipitation method. Herein, we created a positively charged surface containing nanoparticles on electrospun Nylon-6 nanofibers (NFs) to improve the separation and selectivity properties for adsorption of negatively charged protein, namely bovine serum albumin (BSA). The decline in permeate flux was analyzed using the framework of classical blocking models and fitting, demonstrated that the transition of fouling mechanisms was dominated during the filtration process. The standard blocking model provided the best fit of the experimental results during the mid-filtration period. The membrane decorated by NPs containing 4at.% zinc cations not only provided maximum BSA separation but also capable of separating higher amounts of BSA molecules (even after 1h filtration) than the pure Nylon membrane. Protein separation was achieved through this membrane with the incorporation of NPs that had high zeta potential (+5.9±0.2mV) and lower particle area (22,155nm(2)). The developed membrane has great potential to act as a high efficiency membrane for capturing BSA. Copyright © 2015 Elsevier B.V. All rights reserved.

Countercurrent Chromatographic Separation of Proteins Using an Eccentric Coiled Column with Synchronous and Nonsynchronous Type-J Planetary Motions

PubMed Central

SHINOMIYA, Kazufusa; YOSHIDA, Kazunori; TOKURA, Koji; TSUKIDATE, Etsuhiro; YANAGIDAIRA, Kazuhiro; ITO, Yoichiro

2015-01-01

Protein separation was performed using the high-speed counter-current chromatograph (HSCCC) at both synchronous and nonsynchronous type-J planetary motions. The partition efficiency was evaluated with two different column configurations, eccentric coil and toroidal coil, on the separation of a set of stable protein samples including cytochrome C, myoglobin and lysozyme with a polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. Better peak resolution was obtained by the eccentric coil than by the toroidal coil using either lower or upper phase as the mobile phase. The peak resolution was further improved using the eccentric coil by the nonsynchronous type-J planetary motion with the combination of 1066 rpm of column rotation and 1000 rpm of revolution. PMID:25765276

Countercurrent chromatographic separation of proteins using an eccentric coiled column with synchronous and nonsynchronous type-J planetary motions.

PubMed

Shinomiya, Kazufusa; Yoshida, Kazunori; Tokura, Koji; Tsukidate, Etsuhiro; Yanagidaira, Kazuhiro; Ito, Yoichiro

2015-01-01

Protein separation was performed using the high-speed countercurrent chromatograph (HSCCC) at both synchronous and nonsynchronous type-J planetary motions. The partition efficiency was evaluated with two different column configurations, eccentric coil and toroidal coil, on the separation of a set of stable protein samples including cytochrome C, myoglobin and lysozyme with a polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. Better peak resolution was obtained by the eccentric coil than by the toroidal coil using either lower or upper phase as the mobile phase. The peak resolution was further improved using the eccentric coil by the nonsynchronous type-J planetary motion with the combination of 1066 rpm of column rotation and 1000 rpm of revolution.

Separation of biogenic materials by electrophoresis under zero gravity (L-3)

NASA Technical Reports Server (NTRS)

Kuroda, Masao

1993-01-01

Electrophoresis separates electrically charged materials by imposing a voltage between electrodes. Though free-flow electrophoresis is used without carriers such as colloids to separate and purify biogenic materials including biogenic cells and proteins in blood, its resolving power and separation efficiency is very low on Earth due to sedimentation, flotation, and thermal convection caused by the specific gravity differences between separated materials and buffer solutions. The objective of this experiment is to make a comparative study of various electrophoresis conditions on the ground and in zero-gravity in order to ultimately develop a method for separating various important 'vial' components which are difficult to separate on the ground.

Partition Efficiency of High-Pitch Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge and Application to Purification of Various Collagenases with Aqueous-Aqueous Polymer Phase Systems

PubMed Central

Shinomiya, Kazufusa; Kobayashi, Hiroko; Inokuchi, Norio; Nakagomi, Kazuya; Ito, Yoichiro

2010-01-01

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially increased stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate – 6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, V and X derived from Clostridium hystolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample, while collagenase N-2 and S-1 from Streptomyces parvulus subsp. citrinus were eluted with impurities at the solvent front with the upper phase. The collagenase from C. hystolyticum retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities. PMID:21869859

Column-coupling strategies for multidimensional electrophoretic separation techniques.

PubMed

Kler, Pablo A; Sydes, Daniel; Huhn, Carolin

2015-01-01

Multidimensional electrophoretic separations represent one of the most common strategies for dealing with the analysis of complex samples. In recent years we have been witnessing the explosive growth of separation techniques for the analysis of complex samples in applications ranging from life sciences to industry. In this sense, electrophoretic separations offer several strategic advantages such as excellent separation efficiency, different methods with a broad range of separation mechanisms, and low liquid consumption generating less waste effluents and lower costs per analysis, among others. Despite their impressive separation efficiency, multidimensional electrophoretic separations present some drawbacks that have delayed their extensive use: the volumes of the columns, and consequently of the injected sample, are significantly smaller compared to other analytical techniques, thus the coupling interfaces between two separations components must be very efficient in terms of providing geometrical precision with low dead volume. Likewise, very sensitive detection systems are required. Additionally, in electrophoretic separation techniques, the surface properties of the columns play a fundamental role for electroosmosis as well as the unwanted adsorption of proteins or other complex biomolecules. In this sense the requirements for an efficient coupling for electrophoretic separation techniques involve several aspects related to microfluidics and physicochemical interactions of the electrolyte solutions and the solid capillary walls. It is interesting to see how these multidimensional electrophoretic separation techniques have been used jointly with different detection techniques, for intermediate detection as well as for final identification and quantification, particularly important in the case of mass spectrometry. In this work we present a critical review about the different strategies for coupling two or more electrophoretic separation techniques and the different intermediate and final detection methods implemented for such separations.

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Free flow electrophoresis in space shuttle program (biotex)

NASA Astrophysics Data System (ADS)

Hannig, Kurt; Bauer, Johann

In the space shuttle program free flow electrophoresis will be applied for separation of proteins, biopolymers and cells. Proteins are to be separated according to the ``Feldsprung-Gradienten'' procedure by Prof. H. Wagner, University of Saarbruecken, biopolymers are to be separated by the isotachophoresis technique by Prof. Schmitz, University of Muenster and we intend to separate cells in order to increase the efficiency of recovery of hybrid cells after electrofusion performed under microgravity in collaboration with Prof. U. Zimmermann, University of Wuerzburg. There are supposed two ways for reaching this goal: Enrichment of cells before electrofusion may enhance the probability that the cells of interest are immortalized. Separation of cells after electrofusion may help to clone the hybrid cells of interest. Under microgravity, the combination of improved electrophoresis with higher electrofusion rates may provide new possibilities for immortalization of cells. This may be a new way to obtain cellular products, which are physiologically glycosylated.

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

Free-flow zone electrophoresis: a novel approach and scale-up for preparative protein separation.

PubMed

Poggel, M; Melin, T

2001-04-01

Different continuously working free-flow zone electrophoresis (FFZE) chambers have already been developed [1, 2]. All of them deal with the problem of distinctive Joule heating. The resulting temperature gradients cause an unstable density field which leads to thermal convection and thus to an intermixing of the different fractions within the chamber. The most promising and simple approach to stabilize the flow is to build chambers with one very small dimension (e.g., h = 0.5 mm) to assure efficient heat withdrawal. This in turn presents substantial disadvantages, namely limited throughput and restricted scale-up potential. The novel approach combines a simplified design and assembly with the possibility of straightforward scale-up. It still operates with one small dimension (d = 1-2 mm) to handle the Joule heating. Here, however, not the dimension perpendicular to the electric field but the dimension parallel to the electric field (separation distance) is chosen as the smallest dimension. The efficiency of the new device is shown by the separation of bovine serum albumin (BSA) and cytochrome c with an overall protein throughput of up to 1.1 g/h, using a cell with a separation volume of less than 20 mL.

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.

A new non-degradative method to purify glycogen.

PubMed

Tan, Xinle; Sullivan, Mitchell A; Gao, Fei; Li, Shihan; Schulz, Benjamin L; Gilbert, Robert G

2016-08-20

Liver glycogen, a complex branched glucose polymer containing a small amount of protein, is important for maintaining glucose homeostasis (blood-sugar control) in humans. It has recently been found that glycogen molecular structure is impaired in diabetes. Isolating the carbohydrate polymer and any intrinsically-attached protein(s) is an essential prerequisite for studying this structural impairment. This requires an effective, non-degradative and efficient purification method to exclude the many other proteins present in liver. Proteins and glycogen have different ranges of molecular sizes. Despite the plethora of proteins that might still be present in significant abundance after other isolation techniques, SEC (size exclusion chromatography, also known as GPC), which separates by molecular size, should separate those extraneous to glycogen from glycogen with any intrinsically associated protein(s). A novel purification method is developed for this, based on preparative SEC following sucrose gradient centrifugation. Proteomics is used to show that the new method compares favourably with current methods in the literature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Physical stage of photosynthesis charge separation

NASA Astrophysics Data System (ADS)

Yakovlev, A. G.; Shuvalov, V. A.

2016-06-01

An analytical review is given concerning the biophysical aspects of light-driven primary charge separation in photosynthesis reaction centers (RCs) which are special pigment-protein complexes residing in a cell membrane. The primary (physical) stage of charge separation occurs in the pico- and femtosecond ranges and consists of transferring an electron along the active A-branch of pigments. The review presents vast factual material on both the general issues of primary photosynthesis and some more specific topics, including (1) the role of the inactive B-branch of pigments, (2) the effect of the protein environment on the charge separation, and (3) the participation of monomeric bacteriochlorophyll BA in primary electron acceptance. It is shown that the electron transfer and stabilization are strongly influenced by crystallographic water and tyrosine M210 molecules from the nearest environment of BA. A linkage between collective nuclear motions and electron transfer upon charge separation is demonstrated. The nature of the high quantum efficiency of primary charge separation reactions is discussed.

Monolithic poly[(trimethylsilyl-4-methylstyrene)-co- bis(4-vinylbenzyl)dimethylsilane] stationary phases for the fast separation of proteins and oligonucleotides.

PubMed

Jakschitz, Thomas A E; Huck, Christian W; Lubbad, Said; Bonn, Günther K

2007-04-13

In this paper the synthesis, optimisation and application of a silane based monolithic copolymer for the rapid separation of proteins and oligonucleotides is described. The monolith was prepared by thermal initiated in situ copolymerisation of trimethylsilyl-4-methylstyrene (TMSiMS) and bis(4-vinylbenzyl)dimethylsilane (BVBDMSi) in a silanised 200 microm I.D. fused silica column. Different ratios of monomer and crosslinker, as well as different ratios of micro- (toluene) and macro-porogen (2-propanol) were used for optimising the physical properties of the stationary phase regarding separation efficiency. The prepared monolithic stationary phases were characterised by measurement of permeability with different solvents, determination of pore size distribution by mercury intrusion porosimetry (MIP). Morphology was studied by scanning electron microscopy (SEM). Applying optimised conditions, a mixture comprised of five standard proteins ribunuclease A, cytochrome c, alpha-lactalbumine, myoglobine and ovalbumine was separated within 1 min by ion-pair reversed-phase liquid chromatography (IP-RPLC) obtaining half-height peak widths between 1.8 and 2.4 s. Baseline separation of oligonucleotides d(pT)(12-18) was achieved within 1.8 min obtaining half-height peak widths between 3.6 and 5.4 s. The results demonstrate the high potential of this stationary phase for fast separation of high-molecular weight biomolecules such as oligonucleotides and proteins.

Superficially Porous Particles with 1000 Å Pores for Large Biomolecule High Performance Liquid Chromatography and Polymer Size Exclusion Chromatography

PubMed Central

Wagner, Brian M.; Schuster, Stephanie A.; Boyes, Barry E.; Shields, Taylor J.; Miles, William L.; Haynes, Mark J.; Moran, Robert E.; Kirkland, Joseph J.; Schure, Mark R.

2017-01-01

To facilitate mass transport and column efficiency, solutes must have free access to particle pores to facilitate interactions with the stationary phase. To ensure this feature, particles should be used for HPLC separations which have pores sufficiently large to accommodate the solute without restricted diffusion. This paper describes the design and properties of superficially porous (also called Fused-Core®, core shell or porous shell) particles with very large (1000 Å) pores specifically developed for separating very large biomolecules and polymers. Separations of DNA fragments, monoclonal antibodies, large proteins and large polystyrene standards are used to illustrate the utility of these particles for efficient, high-resolution applications. PMID:28213987

Superficially porous particles with 1000Å pores for large biomolecule high performance liquid chromatography and polymer size exclusion chromatography.

PubMed

Wagner, Brian M; Schuster, Stephanie A; Boyes, Barry E; Shields, Taylor J; Miles, William L; Haynes, Mark J; Moran, Robert E; Kirkland, Joseph J; Schure, Mark R

2017-03-17

To facilitate mass transport and column efficiency, solutes must have free access to particle pores to facilitate interactions with the stationary phase. To ensure this feature, particles should be used for HPLC separations which have pores sufficiently large to accommodate the solute without restricted diffusion. This paper describes the design and properties of superficially porous (also called Fused-Core ® , core shell or porous shell) particles with very large (1000Å) pores specifically developed for separating very large biomolecules and polymers. Separations of DNA fragments, monoclonal antibodies, large proteins and large polystyrene standards are used to illustrate the utility of these particles for efficient, high-resolution applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Modular Homogeneous Chromophore–Catalyst Assemblies

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

Mulfort, Karen L.; Utschig, Lisa M.

2016-05-17

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate thatmore » molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.« less

Effects of ozone and peroxone on algal separation via dispersed air flotation.

PubMed

Nguyen, Truc Linh; Lee, D J; Chang, J S; Liu, J C

2013-05-01

Effects of pre-oxidation on algal separation by dispersed air flotation were examined. Ozone (O3) and peroxone (O3 and H2O2) could induce cell lysis, release of intracellular organic matter (IOM), and mineralization of organic substances. Separation efficiency of algal cells improved when pre-oxidized. Total of 76.4% algal cells was separated at 40 mg/L of N-cetyl-N-N-N-trimethylammonium bromide (CTAB), while 95% were separated after 30-min ozonation. Pre-oxidation by ozone and peroxone also enhanced flotation separation efficiency of dissolved organic carbon (DOC), polysaccharide, and protein, in which peroxone process exerted more significantly than O3. Two main mechanisms were involved in flotation separation of unoxidized algal suspension, namely hydrophobic cell surface and cell flocculation resulting from CTAB adsorption. However, flocculation by CTAB was hindered for pre-oxidized algal suspensions. It implied that the compositional changes in extracellular organic matter (EOM) by pre-oxidation were more determined for flotation separation of pre-oxidized cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Highly selective isolation and purification of heme proteins in biological samples using multifunctional magnetic nanospheres.

PubMed

Liu, Yating; Li, Yan; Wei, Yun

2014-12-01

Magnetic particles with suitable surface modification are capable of binding proteins selectively, and magnetic separations have advantages of rapidity, convenience, and high selectivity. In this paper, new magnetic nanoparticles modified with imidazolium ionic liquid (Fe3O4 @SiO2 @ILs) were successfully fabricated. N-Methylimidazolium was immobilized onto silica-coated magnetic nanoparticles via γ-chloropropyl modification as a magnetic nanoadsorbent for heme protein separation. The particle size was about 90 nm without significant aggregation during the preparation process. Hemoglobin as one of heme proteins used in this experiment was compared with other nonheme proteins. It has been found that the magnetic nanoparticles can be used for more rapid, efficient, and specific adsorption of hemoglobin with a binding capacity as high as 5.78 mg/mg. In comparison with other adsorption materials of proteins in the previous reports, Fe3 O4 @SiO2 @ILs magnetic nanoparticles exhibit the excellent performance in isolation of heme proteins with higher binding capacity and selectivity. In addition, a short separation time makes the functionalized nanoparticles suitable for purifying unstable proteins, as well as having other potential applications in a variety of biomedical fields. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

PubMed Central

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.

2014-01-01

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally. PMID:24459184

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

PubMed

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C; Landes, Christy F

2014-02-11

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

An Efficient Method for Co-purification of Eggshell Matrix Proteins OC-17, OC-116, and OCX-36

PubMed Central

2016-01-01

In this study, we improved the eggshell-membrane separation process by separating the shell and membrane with EDTA solution, evaluating effects of three different extraction solutions (acetic acid, EDTA, and phosphate solution), and co-purifying multiple eggshell proteins with two successive ion-exchange chromatography procedures (CM Sepharose Fast Flow and DEAE Sepharose Fast Flow). The recovery and residual rates of eggshell and membrane separated by the modified method with added EDTA solution were 93.88%, 91.15% and 1.01%, 2.87%, respectively. Ovocleidin-116 (OC-116) and ovocalyxin-36 (OCX-36) were obtained by loading 50 mM Na-Hepes, pH 7.5, 2 mM DTT and 350 mM NaCl buffer onto the DEAE-FF column at a flow rate of 1 mL/min, ovocleidin-17 (OC-17) was obtained by loading 100 mM NaCl, 50 mM Tris, pH 8.0 on the CM-FF column at a flow rate of 0.5 mL/min. The purities of OCX-36, OC-17 and OC-116 were 96.82%, 80.15% and 73.22%, and the recovery rates were 55.27%, 53.38% and 36.34%, respectively. Antibacterial activity test suggested that phosphate solution extract exhibited significantly higher activity against the tested bacterial strains than the acetic acid or EDTA extract, probably due to more types of proteins in the extract. These results demonstrate that this separation method is feasible and efficient. PMID:28115888

A novel method for separation of caseins from milk by phosphates precipitation.

PubMed

Yen, Chon-Ho; Lin, Yin-Shen; Tu, Ching-Fu

2015-01-01

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

A colloidal assembly approach to synthesize magnetic porous composite nanoclusters for efficient protein adsorption

NASA Astrophysics Data System (ADS)

Yang, Qi; Lan, Fang; Yi, Qiangying; Wu, Yao; Gu, Zhongwei

2015-10-01

A combination strategy of the inverse emulsion crosslinking approach and the colloidal assembly technique is first proposed to synthesize Fe3O4/histidine composite nanoclusters as new-type magnetic porous nanomaterials. The nanoclusters possess uniform morphology, high magnetic content and excellent protein adsorption capacity, exhibiting their great potential for bio-separation.A combination strategy of the inverse emulsion crosslinking approach and the colloidal assembly technique is first proposed to synthesize Fe3O4/histidine composite nanoclusters as new-type magnetic porous nanomaterials. The nanoclusters possess uniform morphology, high magnetic content and excellent protein adsorption capacity, exhibiting their great potential for bio-separation. Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c5nr05800g

Comparison of twin-cell centrifugal partition chromatographic columns with different cell volume.

PubMed

Goll, Johannes; Audo, Gregoire; Minceva, Mirjana

2015-08-07

Two twin-cell centrifugal partition chromatographic columns (SCPC 250 and SCPE-250-BIO, Armen Instrument, France) with the same column volume but different cell size and number were compared in terms of stationary phase retention and column efficiency. The columns were tested with two types of solvent systems: a commonly used organic solvent based biphasic system from the ARIZONA solvent system family and a polymer/salt based aqueous two phase system (ATPS). The efficiency of the columns was evaluated by pulse injection experiments of two benzenediols (pyrocatechol and hydroquinone) in the case of the ARIZONA system and a protein mixture (myoglobin and lysozyme) in the case of the ATPS. As result of high stationary phase retention, the column with the lower number of larger twin-cells (SCPE-250-BIO) is suitable for protein separations using ATPS. On the other hand, due to higher column efficiency, the column with the greater number of smaller cells (SCPC 250) is superior for batch elution separations performed with standard liquid-liquid chromatography organic solvent based biphasic systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytocompatible and water stable ultrafine protein fibers for tissue engineering

NASA Astrophysics Data System (ADS)

Jiang, Qiuran

This dissertation proposal focuses on the development of cytocompatible and water stable protein ultrafine fibers for tissue engineering. The protein-based ultrafine fibers have the potential to be used for biomedicine, due to their biocompatibility, biodegradability, similarity to natural extracellular matrix (ECM) in physical structure and chemical composition, and superior adsorption properties due to their high surface to volume ratio. However, the current technologies to produce the protein-based ultrafine fibers for biomedical applications still have several problems. For instance, the current electrospinning and phase separation technologies generate scaffolds composed of densely compacted ultrafine fibers, and cells can spread just on the surface of the fiber bulk, and hardly penetrate into the inner sections of scaffolds. Thus, these scaffolds can merely emulate the ECM as a two dimensional basement membrane, but are difficult to mimic the three dimensional ECM stroma. Moreover, the protein-based ultrafine fibers do not possess sufficient water stability and strength for biomedical applications, and need modifications such as crosslinking. However, current crosslinking methods are either high in toxicity or low in crosslinking efficiency. To solve the problems mentioned above, zein, collagen, and gelatin were selected as the raw materials to represent plant proteins, animal proteins, and denatured proteins in this dissertation. A benign solvent system was developed specifically for the fabrication of collagen ultrafine fibers. In addition, the gelatin scaffolds with a loose fibrous structure, high cell-accessibility and cell viability were produced by a novel ultralow concentration phase separation method aiming to simulate the structure of three dimensional (3D) ECM stroma. Non-toxic crosslinking methods using citric acid as the crosslinker were also developed for electrospun or phase separated scaffolds from these three proteins, and proved to be efficient to enhance the strength and water stability of scaffolds. The crosslinked protein scaffolds showed higher cytocompatibility than the polylactic acid scaffolds and the fibers crosslinked by glutaraldehyde. The potential of using these protein-based ultrafine fibers crosslinked by citric acid for tissue engineering has been proved in this dissertation.

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

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Free-solution electrophoretic separations of DNA–drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

PubMed Central

Albrecht, Jennifer Coyne; Kerby, Matthew B.; Niedringhaus, Thomas P.; Lin, Jennifer S.; Wang, Xiaoxiao; Barron, Annelise E.

2012-01-01

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of Free-Solution Conjugate Electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. Single-stranded DNA separations in “gels” have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE’s ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with pHEA-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags. PMID:21500207

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.

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins ☆

PubMed Central

Donnelly, Mark I.; Zhou, Min; Millard, Cynthia Sanville; Clancy, Shonda; Stols, Lucy; Eschenfeldt, William H.; Collart, Frank R.; Joachimiak, Andrzej

2009-01-01

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his6-tag–maltose-binding protein (MBP), intended to facilitate purification and enhance proteins’ solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his6-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his6-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his6-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his6-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his6-tag. PMID:16497515

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

PubMed Central

Borges, Chad R.; Jarvis, Jason W.; Oran, Paul E.; Rogers, Stephen P.; Nelson, Randall W.

2008-01-01

Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID:19137103

Phase separation and the formation of the pyrenoid, a carbon-fixing organelle

NASA Astrophysics Data System (ADS)

Xu, Bin; Freeman Rosenzweig, Elizabeth; Mackinder, Luke; Jonikas, Martin; Wingreen, Ned S.

In the chloroplasts of most algae, the carbon-fixing enzyme Rubisco is concentrated in a non-membrane-bound structure called the pyrenoid, which enables more efficient carbon capture than that of most land plants. In contrast to the long-held assumptions of the field, the pyrenoid matrix is not a solid crystal, but behaves as a phase-separated, liquid-like organelle. In this system, the linker protein EPYC1 is thought to form multivalent specific bonds with Rubisco, and the formation of the pyrenoid occurs via the phase separation of these two associating proteins. Through analytical and numerical studies, we determine a phase diagram for this system. We also show how the length of the linker protein can affect the formation and dissolution of the pyrenoid in an unexpected manner. This new view of the pyrenoid matrix provides important insights into the structure, regulation, and inheritance of pyrenoid. More broadly, our findings give insights into fundamental principles of the architecture and inheritance of liquid-phase organelles.

Process efficiency of casein separation from milk using polymeric spiral-wound microfiltration membranes.

PubMed

Mercier-Bouchard, D; Benoit, S; Doyen, A; Britten, M; Pouliot, Y

2017-11-01

Microfiltration is largely used to separate casein micelles from milk serum proteins (SP) to produce a casein-enriched retentate for cheese making and a permeate enriched in native SP. Skim milk microfiltration is typically performed with ceramic membranes and little information is available about the efficiency of spiral-wound (SW) membranes. We determined the effect of SW membrane pore size (0.1 and 0.2 µm) on milk protein separation in total recirculation mode with a transmembrane pressure gradient to evaluate the separation efficiency of milk proteins and energy consumption after repeated concentration and diafiltration (DF). Results obtained in total recirculation mode demonstrated that pore size diameter had no effect on the permeate flux, but a drastic loss of casein was observed in permeate for the 0.2-µm SW membrane. Concentration-DF experiments (concentration factor of 3.0× with 2 sequential DF) were performed with the optimal 0.1-µm SW membrane. We compared these results to previous data we generated with the 0.1-µm graded permeability (GP) membrane. Whereas casein rejection was similar for both membranes, SP rejection was higher for the 0.1-µm SW membrane (rejection coefficient of 0.75 to 0.79 for the 0.1-µm SW membrane versus 0.46 to 0.49 for the GP membrane). The 0.1-µm SW membrane consumed less energy (0.015-0.024 kWh/kg of permeate collected) than the GP membrane (0.077-0.143 kWh/kg of permeate collected). A techno-economic evaluation led us to conclude that the 0.1-µm SW membranes may represent a better option to concentrate casein for cheese milk; however, the GP membrane has greater permeability and its longer lifetime (about 10 yr) potentially makes it an interesting option. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

PubMed

Corradini, D; Huber, C G; Timperio, A M; Zolla, L

2000-07-21

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

NASA Astrophysics Data System (ADS)

Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

2017-12-01

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

In vitro folding of inclusion body proteins.

PubMed

Rudolph, R; Lilie, H

1996-01-01

Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Linear and Nonlinear Spectroscopic Probing of Solute Interactions with Chemically Modified Silica Surfaces

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

Wirth, Mary J

Solar energy conversion through biology would provide a renewable and nonpolluting abundance of energy. The bacterium Halobacterium salinarum converts solar to electrical energy by virtue of a transmembrane protein, bacteriorhodopsin. This transmembrane protein pumps protons across a nonconducting bilayer upon irradiation with green light. The bacterium evolved to perform this function inefficiently. If we were able to understand this process to engineer this protein for efficiency, then inexpensive energy production could be achieved. There are tens of thousands of different types of halobacteria, giving the opportunity to study different efficiencies and relating these to the protein structures. Technology does notmore » yet exist to perform such screening. The goal of this research is to generate new separation technology that can ultimately enable such screening. This involves creating a method for separating oriented and functional transmembrane proteins that remain in an electrically insulating lipid bilayer, with aqueous solutions on either side of the bilayer. A pH change across the lipid bilayer upon irradiation of a known concentration of proteins would probe function. Differences in proton pumping efficiency for different proteins variants would provide structure-function information for engineering the proteins. A schematic diagram from the original proposal is shown here. The idea is that (a) a lipid bilayer supported on a hydrophilic polymer film will make the bilayer fluid, and (b) applying an electric field will cause electrophoretic migration of the transmembrane proteins. We demonstrated this concept experimentally in a paper that was published just after this new grant period started (Lipid Bilayers on Polyacrylamide Brushes for Inclusion of Membrane Proteins, Emily A. Smith, Jason W. Coym, Scott M. Cowell, Victor J. Hruby, Henry I. Yamamura, Mary J. Wirth, Langmuir, 21, 9644-9650, 2005). The electrophoretic mobility was slow (10{sup -8} cm{sup 2}/Vs), and we project that a two order of magnitude increase would make this a practical tool. We are investigating two ways of improving electrophoretic mobility: better polymer supports, and a novel nanoporous medium that suspends the bilayer over free solution.« less

Production of Mushroom Mycelium as a Protein and Fat Source in Submerged Culture in Medium of Vinasse

PubMed Central

Falanghe, H.

1962-01-01

Of ten mushroom cultures investigated, only Agaricus campestris, Boletus indecisus, and Tricholoma nudum were capable of growing in submerged culture in medium of vinasse with added salts. Higher fermentative efficiencies were found under these conditions than in medium containing molasses or waste sulfite liquor. A. campestris showed a better capacity to produce protein but, since B. indecisus is capable of developing greater mycelium weight, its fermentative efficiencies are comparable. Both microorganisms could be grown in medium of vinasse with greatly varied amounts, producing higher mycelial weight in media with greater vinasse. The capacity of B. indecisus and A. campestris to utilize the noncarbohydrate fraction in total solids, instead of the total carbohydrates when they are in smaller amount, was observed in medium containing vinasse. B. indecisus and A. campestris were easily separated by filtration from the medium, although T. nudum was difficult to separate by this procedure. In experiments with A. campestris, the adaptative capacity of the organism to vinasse was demonstrated. PMID:13962715

Concentration and purification of HIV-1 virions by microfluidic separation of superparamagnetic nanoparticles

PubMed Central

Chen, Grace Dongqing; Alberts, Catharina Johanna

2009-01-01

The low concentration and complex sample matrix of many clinical and environmental viral samples presents a significant challenge in the development of low cost, point-of-care viral assays. To address this problem, we investigated the use of a microfluidic passive magnetic separator combined with on-chip mixer to both purify and concentrate whole particle HIV-1 virions. Virus-containing plasma samples are first mixed to allow specific binding of the viral particles with antibody-conjugated superparamagnetic nanoparticles, and several passive mixer geometries were assessed for their mixing efficiencies. The virus-nanoparticle complexes are then separated from the plasma in a novel magnetic separation chamber, where packed micron-sized ferromagnetic particles serve as high magnetic gradient concentrators for an externally applied magnetic field. Thereafter, a viral lysis buffer was flowed through the chip and the released HIV proteins were assayed off-chip. Viral protein extraction efficiencies of 62% and 45% were achieved at 10uL/min and 30uL/min throughputs respectively. More importantly, an 80-fold concentration was observed for an initial sample volume of 1mL, and a 44-fold concentration for an initial sample volume of 0.5mL. The system is broadly applicable to microscale sample preparation of any viral sample and can be used for nucleic acid extraction as well as 40–80 fold enrichment of target viruses. PMID:19954210

Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

NASA Astrophysics Data System (ADS)

Pietsch, Jessica; Bauer, Johann; Weber, Gerhard; Nissum, Mikkel; Westphal, Kriss; Egli, Marcel; Grosse, Jirka; Schönberger, Johann; Eilles, Christoph; Infanger, Manfred; Grimm, Daniela

2011-11-01

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1 g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11 days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1 g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71 kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

PubMed Central

Ravi, Ayswarya; Guo, Shengchun; Rasala, Beth; Tran, Miller; Mayfield, Stephen; Nikolov, Zivko L.

2018-01-01

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN. PMID:29462927

ATPS: "Aqueous two-phase System" as the "Answer to Protein Separation" for protein-processing Food Industry.

PubMed

Khan, Bilal Muhammad; Liu, Zhi-Cong; Shi, Fu-Lin; Cheong, Kit-Leong; Liu, Yang

2018-06-08

Every individual needs food for its nutritional value and flavor while the economic growth of a nation depends on a thriving profit-generating industry. The food industry caters to both needs in an efficient manner. Proteins can rightly be considered as the driving force behind the overwhelming success of this industry. However, purification of proteins is not an easy undertaking due to their intricate nature while presently employed procedures for this purpose, regrettably, are both costly, and labor- and time-intensive in addition to being unsettling on proteins structural conformity. ATPS has accumulated a lot of interest from the scientific community due to its mild operating conditions, high recovery yield, ease of scaling it up, and its cost-effective and environment friendly nature. This review tries to amass some accounts concerning the utility of ATPS for the separation and purification of proteins. Some auspicious clues in this regard can be witnessed along with a few loopholes which need to be addressed before this technique can truly demonstrate its potential vis-à-vis industrial protein purification. Overall, a polymer - salt (citrates in particular) ATPS with an added inert supplementary salt can be regarded as a better option for purifying proteins.

Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

PubMed

Becker, J Susanne; Mounicou, Sandra; Zoriy, Miroslav V; Becker, J Sabine; Lobinski, Ryszard

2008-09-15

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.

Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

PubMed

Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

2011-03-01

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

Protein electron transfer: Dynamics and statistics

NASA Astrophysics Data System (ADS)

Matyushov, Dmitry V.

2013-07-01

Electron transfer between redox proteins participating in energy chains of biology is required to proceed with high energetic efficiency, minimizing losses of redox energy to heat. Within the standard models of electron transfer, this requirement, combined with the need for unidirectional (preferably activationless) transitions, is translated into the need to minimize the reorganization energy of electron transfer. This design program is, however, unrealistic for proteins whose active sites are typically positioned close to the polar and flexible protein-water interface to allow inter-protein electron tunneling. The high flexibility of the interfacial region makes both the hydration water and the surface protein layer act as highly polar solvents. The reorganization energy, as measured by fluctuations, is not minimized, but rather maximized in this region. Natural systems in fact utilize the broad breadth of interfacial electrostatic fluctuations, but in the ways not anticipated by the standard models based on equilibrium thermodynamics. The combination of the broad spectrum of static fluctuations with their dispersive dynamics offers the mechanism of dynamical freezing (ergodicity breaking) of subsets of nuclear modes on the time of reaction/residence of the electron at a redox cofactor. The separation of time-scales of nuclear modes coupled to electron transfer allows dynamical freezing. In particular, the separation between the relaxation time of electro-elastic fluctuations of the interface and the time of conformational transitions of the protein caused by changing redox state results in dynamical freezing of the latter for sufficiently fast electron transfer. The observable consequence of this dynamical freezing is significantly different reorganization energies describing the curvature at the bottom of electron-transfer free energy surfaces (large) and the distance between their minima (Stokes shift, small). The ratio of the two reorganization energies establishes the parameter by which the energetic efficiency of protein electron transfer is increased relative to the standard expectations, thus minimizing losses of energy to heat. Energetically efficient electron transfer occurs in a chain of conformationally quenched cofactors and is characterized by flattened free energy surfaces, reminiscent of the flat and rugged landscape at the stability basin of a folded protein.

Protein electron transfer: Dynamics and statistics.

PubMed

Matyushov, Dmitry V

2013-07-14

Electron transfer between redox proteins participating in energy chains of biology is required to proceed with high energetic efficiency, minimizing losses of redox energy to heat. Within the standard models of electron transfer, this requirement, combined with the need for unidirectional (preferably activationless) transitions, is translated into the need to minimize the reorganization energy of electron transfer. This design program is, however, unrealistic for proteins whose active sites are typically positioned close to the polar and flexible protein-water interface to allow inter-protein electron tunneling. The high flexibility of the interfacial region makes both the hydration water and the surface protein layer act as highly polar solvents. The reorganization energy, as measured by fluctuations, is not minimized, but rather maximized in this region. Natural systems in fact utilize the broad breadth of interfacial electrostatic fluctuations, but in the ways not anticipated by the standard models based on equilibrium thermodynamics. The combination of the broad spectrum of static fluctuations with their dispersive dynamics offers the mechanism of dynamical freezing (ergodicity breaking) of subsets of nuclear modes on the time of reaction/residence of the electron at a redox cofactor. The separation of time-scales of nuclear modes coupled to electron transfer allows dynamical freezing. In particular, the separation between the relaxation time of electro-elastic fluctuations of the interface and the time of conformational transitions of the protein caused by changing redox state results in dynamical freezing of the latter for sufficiently fast electron transfer. The observable consequence of this dynamical freezing is significantly different reorganization energies describing the curvature at the bottom of electron-transfer free energy surfaces (large) and the distance between their minima (Stokes shift, small). The ratio of the two reorganization energies establishes the parameter by which the energetic efficiency of protein electron transfer is increased relative to the standard expectations, thus minimizing losses of energy to heat. Energetically efficient electron transfer occurs in a chain of conformationally quenched cofactors and is characterized by flattened free energy surfaces, reminiscent of the flat and rugged landscape at the stability basin of a folded protein.

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography.

PubMed

Bakry, R; Stöggl, W M; Hochleitner, E O; Stecher, G; Huck, C W; Bonn, G K

2006-11-03

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.

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.

A simple tool for tubing modification to improve spiral high-speed counter-current chromatography for protein purification

PubMed Central

Ito, Yoichiro; Ma, Xiaofeng; Clary, Robert

2016-01-01

A simple tool is introduced which can modify the shape of tubing to enhance the partition efficiency in high-speed countercurrent chromatography. It consists of a pair of interlocking identical gears, each coaxially holding a pressing wheel to intermittently compress plastic tubing in 0 – 10 mm length at every 1 cm interval. The performance of the processed tubing is examined in protein separation with 1.6 mm ID PTFE tubing intermittently pressed in 3 mm and 10 mm width both at 10 mm intervals at various flow rates and revolution speeds. A series of experiments was performed with a polymer phase system composed of polyethylene glycol and dibasic potassium phosphate each at 12.5% (w/w) in deionized water using three protein samples. Overall results clearly demonstrate that the compressed tubing can yield substantially higher peak resolution than the non-processed tubing. The simple tubing modifier is very useful for separation of proteins with high-speed countercurrent chromatography. PMID:27818942

A simple tool for tubing modification to improve spiral high-speed counter-current chromatography for protein purification.

PubMed

Ito, Yoichiro; Ma, Xiaofeng; Clary, Robert

2016-01-01

A simple tool is introduced which can modify the shape of tubing to enhance the partition efficiency in high-speed countercurrent chromatography. It consists of a pair of interlocking identical gears, each coaxially holding a pressing wheel to intermittently compress plastic tubing in 0 - 10 mm length at every 1 cm interval. The performance of the processed tubing is examined in protein separation with 1.6 mm ID PTFE tubing intermittently pressed in 3 mm and 10 mm width both at 10 mm intervals at various flow rates and revolution speeds. A series of experiments was performed with a polymer phase system composed of polyethylene glycol and dibasic potassium phosphate each at 12.5% (w/w) in deionized water using three protein samples. Overall results clearly demonstrate that the compressed tubing can yield substantially higher peak resolution than the non-processed tubing. The simple tubing modifier is very useful for separation of proteins with high-speed countercurrent chromatography.

Surfing on Protein Waves: Proteophoresis as a Mechanism for Bacterial Genome Partitioning

NASA Astrophysics Data System (ADS)

Walter, J.-C.; Dorignac, J.; Lorman, V.; Rech, J.; Bouet, J.-Y.; Nollmann, M.; Palmeri, J.; Parmeggiani, A.; Geniet, F.

2017-07-01

Efficient bacterial chromosome segregation typically requires the coordinated action of a three-component machinery, fueled by adenosine triphosphate, called the partition complex. We present a phenomenological model accounting for the dynamic activity of this system that is also relevant for the physics of catalytic particles in active environments. The model is obtained by coupling simple linear reaction-diffusion equations with a proteophoresis, or "volumetric" chemophoresis, force field that arises from protein-protein interactions and provides a physically viable mechanism for complex translocation. This minimal description captures most known experimental observations: dynamic oscillations of complex components, complex separation, and subsequent symmetrical positioning. The predictions of our model are in phenomenological agreement with and provide substantial insight into recent experiments. From a nonlinear physics view point, this system explores the active separation of matter at micrometric scales with a dynamical instability between static positioning and traveling wave regimes triggered by the dynamical spontaneous breaking of rotational symmetry.

Preparation of well-controlled three-dimensional skeletal hybrid monoliths via thiol-epoxy click polymerization for highly efficient separation of small molecules in capillary liquid chromatography.

PubMed

Lin, Hui; Chen, Lianfang; Ou, Junjie; Liu, Zhongshan; Wang, Hongwei; Dong, Jing; Zou, Hanfa

2015-10-16

Two kinds of hybrid monoliths were first prepared via thiol-epoxy click polymerization using a multi-epoxy monomer, octaglycidyldimethylsilyl POSS (POSS-epoxy), and two multi-thiols, trimethylolpropanetris(3-mercaptopropionate) (TPTM) and pentaerythritoltetrakis(3-mercaptopropionate) (PTM), respectively, as the precursors. The resulting two hybrid monoliths (assigned as POSS-epoxy-TPTM and POSS-epoxy-PTM) not only possessed high thermal, mechanical and chemical stabilities, but also exhibited well-controlled 3D skeletal microstructure and high efficiency in capillary liquid chromatography (cLC) separation of small molecules. The highest column efficiency reached 182,700N/m (for butylbenzene) on the monolith POSS-epoxy-PTM at the velocity of 0.75mm/s. Furthermore, the hybrid monolith POSS-epoxy-PTM was successfully applied for cLC separations of various samples, not only standard compounds such as alkylbenzenes, PAHs, phenols and dipeptides, as well as intact proteins, but also complex samples of EPA 610 and BSA digest. Copyright © 2015 Elsevier B.V. All rights reserved.

Separation of multiphosphorylated peptide isomers by hydrophilic interaction chromatography on an aminopropyl phase.

PubMed

Singer, David; Kuhlmann, Julia; Muschket, Matthias; Hoffmann, Ralf

2010-08-01

The separation of isomeric phosphorylated peptides is challenging and often impossible for multiphosphorylated isomers using chromatographic and capillary electrophoretic methods. In this study we investigated the separation of a set of single-, double-, and triple-phosphorylated peptides (corresponding to the human tau protein) by ion-pair reversed-phase chromatography (IP-RPC) and hydrophilic interaction chromatography (HILIC). In HILIC both hydroxyl and aminopropyl stationary phases were tested with aqueous acetonitrile in order to assess their separation efficiency. The hydroxyl phase separated the phosphopeptides very well from the unphosphorylated analogue, while on the aminopropyl phase even isomeric phosphopeptides attained baseline separation. Thus, up to seven phosphorylated versions of a given tau domain were separated. Furthermore, the low concentration of an acidic ammonium formate buffer allowed an online analysis with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to be conducted, enabling peptide sequencing and identification of phosphorylation sites.

De-pulping and Seed Separation from Tumba ( Citrullus colocynthis) Fruit

NASA Astrophysics Data System (ADS)

Mudgal, Vishvambhar Dayal

2017-09-01

Tumba ( Citrullus colocynthis) contains spongy pulp in which seeds are embedded unevenly. Seeds contain about 26% fats and 13% protein. The process of seed separation is highly time consuming and labour intensive. Two weeks are required to separate its seeds with traditional methods. The developed prototype, for separating tumba seeds, mainly consists of chopper, de-pulping screw, barrel assembly and seed separation unit. The de-pulping screw and barrel assembly was divided in two sections i.e. conveying (feeding zone) and compression sections (de-pulping zone). The performance of developed machine was evaluated at different screw speed in the range of 40-100 rpm. Maximum pulp removal efficiency of 78.1% was achieved with screw speed of 60 rpm. Seed separation from the pulp was carried out by adding different chemicals. Use of sodium hydroxide and potassium hydroxide produced seed separation up to 99%.

Nonlinear multi-photon laser wave-mixing optical detection in microarrays and microchips for ultrasensitive detection and separation of biomarkers for cancer and neurodegenerative diseases

NASA Astrophysics Data System (ADS)

Iwabuchi, Manna; Hetu, Marcel; Maxwell, Eric; Pradel, Jean S.; Ramos, Sashary; Tong, William G.

2015-09-01

Multi-photon degenerate four-wave mixing is demonstrated as an ultrasensitive absorption-based optical method for detection, separation and identification of biomarker proteins in the development of early diagnostic methods for HIV- 1, cancer and neurodegenerative diseases using compact, portable microarrays and capillary- or microchip-based chemical separation systems that offer high chemical specificity levels. The wave-mixing signal has a quadratic dependence on concentration, and hence, it allows more reliable monitoring of smaller changes in analyte properties. Our wave-mixing detection sensitivity is comparable or better than those of current methods including enzyme-linked immunoassay for clinical diagnostic and screening. Detection sensitivity is excellent since the wave-mixing signal is a coherent laser-like beam that can be collected with virtually 100% collection efficiency with high S/N. Our analysis time is short (1-15 minutes) for molecular weight-based protein separation as compared to that of a conventional separation technique, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When ultrasensitive wavemixing detection is paired with high-resolution capillary- or microchip-based separation systems, biomarkers can be separated and identified at the zepto- and yocto-mole levels for a wide range of analytes. Specific analytes can be captured in a microchannel through the use of antibody-antigen interactions that provide better chemical specificity as compared to size-based separation alone. The technique can also be combined with immune-precipitation and a multichannel capillary array for high-throughput analysis of more complex protein samples. Wave mixing allows the use of chromophores and absorption-modifying tags, in addition to conventional fluorophores, for online detection of immunecomplexes related to cancer.

DURIP: Electrokinetic Injection and Separation System for Analysis of Protein and Peptide Transport, Adsorption and Kinetics Instrumentation Proposal

DTIC Science & Technology

2015-03-18

both the electric double layer that forms at a solid-liquid interface as well as the biomolecules themselves, we can harness the coupled physics of...the biomolecules themselves, we can harness the coupled physics of complex biological fluids in nanofluidic channels towards unique, efficient

Comparison of different immobilized systems in the removal of peanut allergens from peanut extracts.

USDA-ARS?s Scientific Manuscript database

The objective of this study was to determine which of the magnetic-bead systems (Ca2+, Fe3+, caffeic acid, hydrophobic) would bind and separate peanut allergens from other proteins in a peanut extract more efficiently. Commercial Ca2+ and hydrophobic magnetic beads, and caffeic-beads (prepared by at...

SAIDE: A Semi-Automated Interface for Hydrogen/Deuterium Exchange Mass Spectrometry.

PubMed

Villar, Maria T; Miller, Danny E; Fenton, Aron W; Artigues, Antonio

2010-01-01

Deuterium/hydrogen exchange in combination with mass spectrometry (DH MS) is a sensitive technique for detection of changes in protein conformation and dynamics. Since temperature, pH and timing control are the key elements for reliable and efficient measurement of hydrogen/deuterium content in proteins and peptides, we have developed a small, semiautomatic interface for deuterium exchange that interfaces the HPLC pumps with a mass spectrometer. This interface is relatively inexpensive to build, and provides efficient temperature and timing control in all stages of enzyme digestion, HPLC separation and mass analysis of the resulting peptides. We have tested this system with a series of standard tryptic peptides reconstituted in a solvent containing increasing concentration of deuterium. Our results demonstrate the use of this interface results in minimal loss of deuterium due to back exchange during HPLC desalting and separation. For peptides reconstituted in a buffer containing 100% deuterium, and assuming that all amide linkages have exchanged hydrogen with deuterium, the maximum loss of deuterium content is only 17% of the label, indicating the loss of only one deuterium molecule per peptide.

SAIDE: A Semi-Automated Interface for Hydrogen/Deuterium Exchange Mass Spectrometry

PubMed Central

Villar, Maria T.; Miller, Danny E.; Fenton, Aron W.; Artigues, Antonio

2011-01-01

Deuterium/hydrogen exchange in combination with mass spectrometry (DH MS) is a sensitive technique for detection of changes in protein conformation and dynamics. Since temperature, pH and timing control are the key elements for reliable and efficient measurement of hydrogen/deuterium content in proteins and peptides, we have developed a small, semiautomatic interface for deuterium exchange that interfaces the HPLC pumps with a mass spectrometer. This interface is relatively inexpensive to build, and provides efficient temperature and timing control in all stages of enzyme digestion, HPLC separation and mass analysis of the resulting peptides. We have tested this system with a series of standard tryptic peptides reconstituted in a solvent containing increasing concentration of deuterium. Our results demonstrate the use of this interface results in minimal loss of deuterium due to back exchange during HPLC desalting and separation. For peptides reconstituted in a buffer containing 100% deuterium, and assuming that all amide linkages have exchanged hydrogen with deuterium, the maximum loss of deuterium content is only 17% of the label, indicating the loss of only one deuterium molecule per peptide. PMID:25309638

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Preparation of quaternary amine monolithic column for strong anion-exchange chromatography and its application to the separation of Enterovirus 71.

PubMed

Gu, Huimin; Yin, Dezhong; Ren, Jie; Zhang, Baoliang; Zhang, Qiuyu

2016-10-15

Large size virion is unable to diffuse into pores of conventional porous chromatography particles. Therefore, separation of virion by conventional column-packing materials is not quite efficient. To solve this problem, a monolithic column with large convective pores and quaternary amine groups was prepared and was applied to separate Enterovirus 71 (EV71, ≈5700-6000kDa). Cross-section, pore structure, hydrodynamic performance, adsorption property and dynamic binding capacity of prepared monolithic column were determined. Double-pore structures, macropore at 2472nm and mesopore at 5-60nm, were formed. The porosity was up to 63.3%, which enable higher permeability and lower back pressure of the monolithic column than commercial UNO™ Q1 column. Based on the breakthrough curves, the loading capacity of bovine serum albumin was calculated to be 42.0mg per column. In addition, prepared quaternary amine monolithic column was proved to be suitable for the separation of protein mixture by strong anion-exchange chromatography. As a practical application, prepared monolith column presents excellent performance to the separation of EV71 from virus-proteins mixture. Copyright © 2016 Elsevier B.V. All rights reserved.

Fractionation of Exosomes and DNA using Size-Based Separation at the Nanoscale

NASA Astrophysics Data System (ADS)

Wunsch, Benjamin; Smith, Joshua; Wang, Chao; Gifford, Stacey; Brink, Markus; Bruce, Robert; Solovitzky, Gustavo; Austin, Robert; Astier, Yann

Exosomes, a key target of ``liquid biopsies'', are nano-vesicles found in nearly all biological fluids. Exosomes are secreted by eukaryotic and prokaryotic cells alike, and contain information about their originating cells, including surface proteins, cytoplasmic proteins, and nucleic acids. One challenge in studying exosome morphology is the difficulty of sorting exosomes by size and surface markers. Common separation techniques for exosomes include ultracentrifugation and ultrafiltration, for preparation of large volume samples, but these techniques often show contamination and significant heterogeneity between preparations. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood; however, the DLD technology has never been translated to the true nanoscale, where it could function on bio-colloids such as exosomes. We have fabricated nanoscale DLD (nanoDLD) arrays capable of rapidly sorting colloids down to 20 nm in continuous flow, and demonstrated size sorting of individual exosome vesicles and dsDNA polymers, opening the potential for on-chip biomolecule separation and diagnosti

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

Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects

NASA Astrophysics Data System (ADS)

Tripathi, Siddhartha; Kumar, Y. V. Balavarun; Agrawal, Amit; Prabhakar, Amit; Joshi, Suhas S.

2016-06-01

In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 μm such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations.

Separation efficiency of free-solution conjugated electrophoresis with drag-tags incorporating a synthetic amino acid.

PubMed

Seo, Kyung-Ho; Chu, Hun-Su; Yoo, Tae Hyeon; Lee, Sun-Gu; Won, Jong-In

2016-03-01

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge-to-friction ratio in free solution, DNA fragments cannot be separated by size. However, size-based separation of DNA is possible in free-solution conjugate electrophoresis (FSCE) if a "drag-tag" is attached to DNA fragments because the tag breaks the linear charge-to-friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free-solution conjugated electrophoresis, generation of a monodisperse drag-tag and identification of a strong, site-specific conjugation method between a DNA fragment and a drag-tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin-like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site-specific conjugation, a methionine residue in drag-tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Preparation and evaluation of a hydrophilic interaction and cation-exchange chromatography stationary phase modified with 2-methacryloyloxyethyl phosphorylcholine.

PubMed

Xiong, Caifeng; Yuan, Jie; Wang, Zhiying; Wang, Siyao; Yuan, Chenchen; Wang, Lili

2018-04-20

In this work, 2-methacryloyloxyethyl phosphorylcholine (MPC) was used as a ligand to prepare a novel mixed-mode chromatography (MMC) stationary phase by the thiol-ene click reaction onto silica (MPC-silica). It was found that this MPC-silica showed the retention characteristics of hydrophilic interaction chromatography (HILIC) and weak cation exchange chromatography (WCX) under suitable mobile phase conditions. In detail, acidic and basic hydrophilic compounds and puerarin from pueraria were separated quickly with HILIC mode. Meanwhile, six standard proteins were allowed to reach baseline separation in WCX mode, and protein separation from egg white was also achieved with this mode. In addition, reduced/denatured lysozyme could be refolded with the MPC-silica column. In the meantime, the MPC-silica has been applied for refolding with simultaneous purification of recombinant human Delta-like1-RGD (rhDll1-RGD) expressed in Escherichia coli. The results show that the mass recovery and purity of rhDll1-RGD could reach 63.4% and 97% by one step, respectively. Furthermore, the reporter assay results demonstrated that refolded with simultaneously purified rhDll1-RGD could efficiently activate the signalling pathway in a dose-dependent manner. In general, this MPC-silica has good resolution and selectivity in the separation of polar compounds and protein samples in different high-performance liquid chromatography (HPLC) modes, and it successfully achieved refolding with simultaneous purification of denatured protein. Copyright © 2018 Elsevier B.V. All rights reserved.

Method and apparatus for the collection, storage, and real time analysis of blood and other bodily fluids

NASA Technical Reports Server (NTRS)

Whitson, Peggy A. (Inventor); Clift, Vaughan L. (Inventor)

1994-01-01

The present invention provides a simple, portable, relatively inexpensive apparatus for accurately and efficiently collecting, separating, testing, and even storing between about 1-20 ml, preferably about 1-10 ml, of blood or other bodily fluid in situ. The apparatus includes a collection chamber bounded on its sides by an opening in a sheet of material, preferably clear plastic, abutting a filter card. The filter card is made of fibrous material, preferably less than about a millimeter thick, having an average pore size of less than about 3 microns. Preferably, the fibers are glass and the fibrous material has an average pore size of about 1 micron. The fibrous material is treated with a carbohydrate/protein mixture which contains between about 1-40 percent wt/vol carbohydrate and about 0.1-15 percent wt/vol nonspecific protein, preferably between about 10-20 percent carbohydrate and about 5-8 percent protein. A preferred carbohydrate/protein mixture comprises about 10 percent mannitol and about 6 percent albumin. The blood or other fluid moves through the filter card by capillary action aided by an absorbent matrix with a high Klemm factor which abuts the filter card. The absorbent matrix and/or filter card can be treated with a wide spectrum of test reagents. The speed, cleanliness, and efficiency of the separation process can be altered by: (a) changing the absolute concentration of the carbohydrate/protein mixture; (b) applying positive or negative pressure to one side of the filter; and/or (c) varying the relative density and pore size of the filter card and absorbent matrix.

Complex coacervation of supercharged proteins with polyelectrolytes.

PubMed

Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

2016-04-21

Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

Solute removal capacity of high cut-off membrane plasma separators.

PubMed

Ohkubo, Atsushi; Kurashima, Naoki; Nakamura, Ayako; Miyamoto, Satoko; Iimori, Soichiro; Rai, Tatemitsu

2013-10-01

In vitro blood filtration was performed by a closed circuit using high cut-off membrane plasma separators, EVACURE EC-2A10 (EC-2A) and EVACURE EC-4A10 (EC-4A). Samples were obtained from sampling sites before the plasma separator, after each plasma separator, and from the ultrafiltrate of each separator. The sieving coefficient (S.C.) of total protein (TP), albumin (Alb), IgG, interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), fibrinogen (Fib), antithrombin III (AT-III), and coagulation factor XIII (FXIII) were calculated. The S.C. of each solute using EC-2A and EC-A4 were as follows; TP: 0.25 and 0.56, Alb: 0.32 and 0.73, IgG: 0.16 and 0.50, IL-6:0.73 and 0.95, IL-8:0.85 and 0.82, TNF-α: 1.07 and 0.99, Fib: 0 and 0, FXIII: 0.07 and 0.17, respectively. When compared with the conventional type of membrane plasma separators, EVACURE could efficiently remove cytokines while retaining coagulation factors such as fibrinogen. Moreover, EC-2A prevented protein loss, whereas EC-4A could remove approximately 50% of IgG. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

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

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers*

PubMed Central

Faries, Kaitlyn M.; Kressel, Lucas L.; Wander, Marc J.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine; Hanson, Deborah K.

2012-01-01

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization. PMID:22247556

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

PubMed Central

Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

2016-01-01

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

Exploring the protein folding free energy landscape: coupling replica exchange method with P3ME/RESPA algorithm.

PubMed

Zhou, Ruhong

2004-05-01

A highly parallel replica exchange method (REM) that couples with a newly developed molecular dynamics algorithm particle-particle particle-mesh Ewald (P3ME)/RESPA has been proposed for efficient sampling of protein folding free energy landscape. The algorithm is then applied to two separate protein systems, beta-hairpin and a designed protein Trp-cage. The all-atom OPLSAA force field with an explicit solvent model is used for both protein folding simulations. Up to 64 replicas of solvated protein systems are simulated in parallel over a wide range of temperatures. The combined trajectories in temperature and configurational space allow a replica to overcome free energy barriers present at low temperatures. These large scale simulations reveal detailed results on folding mechanisms, intermediate state structures, thermodynamic properties and the temperature dependences for both protein systems.

Major intrinsic proteins in biomimetic membranes.

PubMed

Nielsen, Claus Hélix

2010-01-01

Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport contribution from both protein and biomimetic support matrix. Also the biomimetic matrix must be encapsulated in order to protect it and make it sufficiently stable in a final application. Here, I specifically discuss the feasibility of developing osmotic biomimetic MIP membranes, but the technical issues are of general concern in the design ofbiomimetic membranes capable of supporting selective transmembrane fluxes.

Evaluation of effects of pH and ionic strength on colloidal stability of IgG solutions by PEG-induced liquid-liquid phase separation.

PubMed

Thompson, Ronald W; Latypov, Ramil F; Wang, Ying; Lomakin, Aleksey; Meyer, Julie A; Vunnum, Suresh; Benedek, George B

2016-11-14

Colloidal stability of IgG antibody solutions is important for pharmaceutical and medicinal applications. Solution pH and ionic strength are two key factors that affect the colloidal stability of protein solutions. In this work, we use a method based on the PEG-induced liquid-liquid phase separation to examine the effects of pH and ionic strength on the colloidal stability of IgG solutions. We found that at high ionic strength (≥0.25M), the colloidal stability of most of our IgGs is insensitive to pH, and at low ionic strength (≤0.15M), all IgG solutions are much more stable at pH 5 than at pH 7. In addition, the PEG-induced depletion force is less efficient in causing phase separation at pH 5 than at pH 7. In contrast to the native inter-protein interaction of IgGs, the effect of depletion force on phase separation of the antibody solutions is insensitive to ionic strength. Our results suggest that the long-range electrostatic inter-protein repulsion at low ionic strength stabilizes the IgG solutions at low pH. At high ionic strength, the short-range electrostatic interactions do not make a significant contribution to the colloidal stability for most IgGs with a few exceptions. The weaker effect of depletion force at lower pH indicates a reduction of protein concentration in the condensed phase. This work advances our basic understanding of the colloidal stability of IgG solutions and also introduces a practical approach to measuring protein colloidal stability under various solution conditions.

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

PubMed

Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

2011-11-01

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics.

PubMed

Haataja, Tatu J K; Koski, M Kristian; Hiltunen, J Kalervo; Glumoff, Tuomo

2011-05-01

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

Determination of Conjugation Efficiency of Antibodies and Proteins to the Superparamagnetic Iron Oxide Nanoparticles by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

NASA Astrophysics Data System (ADS)

Wang, Fu-Hua; Yoshitake, Takashi; Kim, Do-Kyung; Muhammed, Mamoun; Bjelke, Börje; Kehr, Jan

2003-04-01

The method based on capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) was developed for determination of magnetic iron oxide nanoparticles (hydrodynamic diameters of 100 nm) functionalized with molecules containing primary amino groups. The magnetic nanoparticles with carboxylic or aminopropyl-trimethoxysilane groups at their surface were conjugated to the model proteins (bovine serum albumin, BSA; streptavidin or goat anti-rabbit immunoglobulin G, IgG) using carbodiimide as a zero-length cross-linker. The nanoparticle-protein conjugates (hydrodynamic diameter 163-194 nm) were derivatized with naphthalene-2,3-dicarboxaldehyde reagent and separated by CE/LIF with a helium-cadmium laser (excitation at 442 nm, emission at 488 nm). The separations were carried out by using a fused-silica capillary (effective length 48 cm, inner diameter 75 um) and 100 mM sodium borate buffer (pH 9.2), the potential was 30 kV. The detection limit for BSA-conjugate was 1.3 pg/10 nl, i.e. about 20 amol. The present method provides an efficient and fast tool for sensitive determination of the efficacy of biomolecular functionalization of magnetic nanoparticles. The CE/LIF technique requires only negligible sample volumes for analysis, which is especially suitable for controlling the process of preparation of functionalized nanoparticles with unique properties aimed to be used for diagnostic or therapeutic purposes.

DEVELOPMENT OF AN AFFINITY SILICA MONOLITH CONTAINING HUMAN SERUM ALBUMIN FOR CHIRAL SEPARATIONS

PubMed Central

Mallik, Rangan; Hage, David S.

2008-01-01

An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3- to 2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: D/L-tryptophan and R/S-warfarin. The separation of R- and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase. PMID:17475436

M13 bacteriophage-activated superparamagnetic beads for affinity separation.

PubMed

Muzard, Julien; Platt, Mark; Lee, Gil U

2012-08-06

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ki-67 acts as a biological surfactant to disperse mitotic chromosomes

PubMed Central

Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z.; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A.; Gerlich, Daniel W.

2016-01-01

Summary Eukaryotic genomes are partitioned into chromosomes, which during mitosis form compact and spatially well-separated mechanical bodies1–3.This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at the chromosome surface3,5,6, the molecular and biophysical basis of mitotic chromosome individuality have remained unclear. We report that Ki-67, a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of Ki-67 is not confined within a specific protein domain but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrical barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-color labeling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic for polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery and suggests that natural proteins can function as surfactants in intracellular compartmentalization. PMID:27362226

Facilitated transport of small molecules and ions for energy-efficient membranes.

PubMed

Li, Yifan; Wang, Shaofei; He, Guangwei; Wu, Hong; Pan, Fusheng; Jiang, Zhongyi

2015-01-07

In nature, the biological membrane can selectively transport essential small molecules/ions through facilitated diffusion via carrier proteins. Intrigued by this phenomenon and principle, membrane researchers have successfully employed synthetic carriers and carrier-mediated reversible reactions to enhance the separation performance of synthetic membranes. However, the existing facilitated transport membranes as well as the relevant facilitated transport theories have scarcely been comprehensively reviewed in the literature. This tutorial review primarily covers the two aspects of facilitated transport theories: carrier-mediated transport mechanisms and facilitated transport chemistries, including the design and fabrication of facilitated transport membranes. The applications of facilitated transport membranes in energy-intensive membrane processes (gas separation, pervaporation, and proton exchange membrane fuel cells) have also been discussed. Hopefully, this review will provide guidelines for the future research and development of facilitated transport membranes with high energy efficiency.

A green deep eutectic solvent-based aqueous two-phase system for protein extracting.

PubMed

Xu, Kaijia; Wang, Yuzhi; Huang, Yanhua; Li, Na; Wen, Qian

2015-03-15

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n=3), 1.6057% (n=3) and 1.6132% (n=3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV-vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES-protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects

PubMed Central

Tripathi, Siddhartha; Kumar, Y. V. BalaVarun; Agrawal, Amit; Prabhakar, Amit; Joshi, Suhas S.

2016-01-01

In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 μm; such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations. PMID:27279146

Capillary trap column with strong cation-exchange monolith for automated shotgun proteome analysis.

PubMed

Wang, Fangjun; Dong, Jing; Jiang, Xiaogang; Ye, Mingliang; Zou, Hanfa

2007-09-01

A 150 microm internal diameter capillary monolithic column with a strong cation-exchange stationary phase was prepared by direct in situ polymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent consisting dimethylsulfoxide, dodecanol, and N,N'-dimethylformamide. This phosphate monolithic column exhibits higher dynamic binding capacity, faster kinetic adsorption of peptides, and more than 10 times higher permeability than the column packed with commercially available strong cation-exchange particles. It was applied as a trap column in a nanoflow liquid chromatography-tandem mass spectrometry system for automated sample injection and online multidimensional separation. It was observed that the sample could be loaded at a flow rate as high as 40 microL/min with a back pressure of approximately 1300 psi and without compromising the separation efficiency. Because of its good orthogonality to the reversed phase separation mechanism, the phosphate monolithic trap column was coupled with a reversed-phase column for online multidimensional separation of 19 microg of the tryptic digest of yeast proteins. A total of 1522 distinct proteins were identified from 5608 unique peptides (total of 54,780 peptides) at the false positive rate only 0.46%.

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.

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

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

Statistical-thermodynamic model for light scattering from eye lens protein mixtures

NASA Astrophysics Data System (ADS)

Bell, Michael M.; Ross, David S.; Bautista, Maurino P.; Shahmohamad, Hossein; Langner, Andreas; Hamilton, John F.; Lahnovych, Carrie N.; Thurston, George M.

2017-02-01

We model light-scattering cross sections of concentrated aqueous mixtures of the bovine eye lens proteins γB- and α-crystallin by adapting a statistical-thermodynamic model of mixtures of spheres with short-range attractions. The model reproduces measured static light scattering cross sections, or Rayleigh ratios, of γB-α mixtures from dilute concentrations where light scattering intensity depends on molecular weights and virial coefficients, to realistically high concentration protein mixtures like those of the lens. The model relates γB-γB and γB-α attraction strengths and the γB-α size ratio to the free energy curvatures that set light scattering efficiency in tandem with protein refractive index increments. The model includes (i) hard-sphere α-α interactions, which create short-range order and transparency at high protein concentrations, (ii) short-range attractive plus hard-core γ-γ interactions, which produce intense light scattering and liquid-liquid phase separation in aqueous γ-crystallin solutions, and (iii) short-range attractive plus hard-core γ-α interactions, which strongly influence highly non-additive light scattering and phase separation in concentrated γ-α mixtures. The model reveals a new lens transparency mechanism, that prominent equilibrium composition fluctuations can be perpendicular to the refractive index gradient. The model reproduces the concave-up dependence of the Rayleigh ratio on α/γ composition at high concentrations, its concave-down nature at intermediate concentrations, non-monotonic dependence of light scattering on γ-α attraction strength, and more intricate, temperature-dependent features. We analytically compute the mixed virial series for light scattering efficiency through third order for the sticky-sphere mixture, and find that the full model represents the available light scattering data at concentrations several times those where the second and third mixed virial contributions fail. The model indicates that increased γ-γ attraction can raise γ-α mixture light scattering far more than it does for solutions of γ-crystallin alone, and can produce marked turbidity tens of degrees celsius above liquid-liquid separation.

Preparation of imprinted cryogel cartridge for chiral separation of l-phenylalanine.

PubMed

Akgönüllü, Semra; Yavuz, Handan; Denizli, Adil

2017-06-01

l-Phe-imprinted cryogel cartridge was prepared for the chiral separation of l-Phe. N-Methacryloyl l-phenylalanine (MAPA) was used as a functional monomer for complexing with l-Phe. The selectivity of the membranes was investigated by using d-Phe, l-Trp, and d-Trp as competitor molecules. The PHEMAPA-l-Trp membranes were 6.4, 4.3, and 5.5 times more selective for l-Phe than d-Phe, l-Trp, and d-Trp, respectively. The PHEMAPA-l-Phe cryogel cartridge was incorporated into the fast protein liquid chromatography (FPLC) equipment and was able to separate D,l-Phe racemic mixture efficiently. The PHEMAPA-l-Phe membranes were shown to be reusable many times without significant loss of the adsorption capacity.

Efficiency and protective effect of encapsulation of milk immunoglobulin G in multiple emulsion.

PubMed

Chen, C C; Tu, Y Y; Chang, H M

1999-02-01

Milk immunoglobulin G (IgG), separated with protein G affinity chromatography, and IgG in colostral whey were encapsulated by 0.5% (w/v) of Tween 80, sucrose stearate, or soy protein, which were used as secondary emulsifiers in the water in oil in water type multiple emulsion. The residual contents of separated IgG and IgG in colostral whey, ranging from 58.7 to 49.7% and from 13.2 to 21.3%, respectively, in the inner water phase (water phase surrounded by oil phase) with emulsifiers were determined by ELISA. However, the emulsion stability decreased after 24 h, and the residual IgG content in the inner water phase was lowered. Encapsulation of IgG in the multiple emulsion increased the stability of separated IgG against acid (pH 2.0) and alkali (pH 12.0) by 21-56% and 33-62%, respectively, depending on the emulsifier used. Moreover, multiple emulsion also provided a remarkable protective effect on separated IgG stability against proteases. The residual contents of separated IgG in multiple emulsion, using Tween 80 as secondary emulsifier, incubated for 2 h with pepsin (pH 2.0) and trypsin and chymotrypsin (pH 7.6) (enzyme/substrate = 1/20) were 35.4, 72.5, and 82.3%, whereas those of separated IgG in enzyme solution were only 7.2, 33. 1, and 35.2%, respectively. However, the separated IgG loss during the preparation of multiple emulsion was almost 41-50%.

Early bacteriopheophytin reduction in charge separation in reaction centers of Rhodobacter sphaeroides.

PubMed

Zhu, Jingyi; van Stokkum, Ivo H M; Paparelli, Laura; Jones, Michael R; Groot, Marie Louise

2013-06-04

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between BA and HA, and the effect of protein conformation on the electron transfer rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

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.

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Monolithic column based on a poly(glycidyl methacrylate-co-4-vinylphenylboronic acid-co-ethylene dimethacrylate) copolymer for capillary liquid chromatography of small molecules and proteins.

PubMed

Lin, Zian; Huang, Hui; Sun, Xiaobo; Lin, Yao; Zhang, Lan; Chen, Guonan

2012-07-13

A new polymer monolith with three modes of reverse-phase, hydrophilic and cation-exchange interaction was synthesized in 100 μm i.d. fused-silica capillary by in situ polymerization procedure. The pre-polymerization mixture consisted of glycidyl methacrylate (GMA) and 4-vinylphenylboronic acid (VPBA) as bifunctional monomers, ethylene dimethacrylate (EDMA) as crosslinker, 1,4-butanediol (BDO) and diethylene glycol (DEG) as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The resulting poly(GMA-co-VPBA-co-EDMA) monolith showed a relatively homogeneous monolithic structure, good permeability and mechanical stability. Different ratios of monomers and porogens were used for optimizing the properties of monolithic column. The column performance was assessed by the separation of a series of neutral solutes, charge solutes, phenols and anilines. Compared with poly(GMA-co-EDMA) monolith, the proposed monolith exhibited more flexible adjustment of selectivity in terms of hydrophobic, hydrophilic, as well as cation-exchange interaction in the same chromatographic conditions. High column efficiencies for benzene derivatives with 70,000-102,000 theoretical plates/m could be obtained at a linear velocity of 0.265 mm/s. The run-to-run, column-to-column, and batch-to-batch repeatabilities of the retention times were less than 8.23%. Additionally, the purposed monolith was also applied to efficient separation of alkaloids and proteins for demonstrating its potential in biomolecule separation. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Isolation of tissue layers in hermatypic corals by N-acetylcysteine: morphological and proteomic examinations

NASA Astrophysics Data System (ADS)

Peng, S.-E.; Luo, Y.-J.; Huang, H.-J.; Lee, I.-T.; Hou, L.-S.; Chen, W.-N. U.; Fang, L.-S.; Chen, C.-S.

2008-03-01

Corals are diploblastic in body pattern and include two tissue layers, the epidermis and gastrodermis, interconnected by an acellular matrix mesoglea. During development, cells in these tissue layers differentiate morphologically and functionally. In most hermatypic corals, the gastrodermis further develops an ability to associate with microalgae dinoflagellates. This endosymbiosis occurs inside specific host gastrodermal cells, and its mechanism still remains unclear notwithstanding decades of research. The delay in progress is partly due to the difficulty in separating the gastrodermis and its symbionts from the epidermis for detailed cellular and biochemical investigations. The present study reports a simple method to separate these two tissue layers in hermatypic corals using the reducing agent, N-acetylcysteine (NAC). Efficient tissue and proteomic isolations are demonstrated by microscopy and two-dimensional SDS polyacrylamide gel electrophoresis (2D SDS-PAGE). The NAC treatment was able to separate tissue layers without inducing protein degradation. Furthermore, the sensitivity of protein detection greatly increases in the isolated tissue layers. The application of the present technique provides future research on endosymbiosis and coral development with a tool for higher accuracy and sensitivity.

Detection and quantification of cocaine and benzoylecgonine in meconium using solid phase extraction and UPLC/MS/MS.

PubMed

Gunn, Josh; Kriger, Scott; Terrell, Andrea R

2010-01-01

The simultaneous determination and quantification of cocaine and its major metabolite, benzoylecgonine, in meconium using UPLC-MS/MS is described. Ultra-performance liquid chromatography (UPLC) is an emerging analytical technique which draws upon the principles of chromatography to run separations at higher flow rates for increased speed, while simultaneously achieving superior resolution and sensitivity. Extraction of cocaine and benzoylecgonine from the homogenized meconium matrix was achieved with a preliminary protein precipitation or protein 'crash' employing cold acetonitrile, followed by a mixed mode solid phase extraction (SPE). Following elution from the SPE cartridge, eluents were dried down under nitrogen, reconstituted in 200 microL of DI water:acetonitrile (ACN) (75:25), and injected onto the UPLC/MS/MS for analysis. The increased speed and separation efficiency afforded by UPLC, allowed for the separation and subsequent quantification of both analytes in less than 2 min. Analytes were quantified using multiple reaction monitoring (MRM) and six-point calibration curves constructed in negative blood. Limits of detection for both analytes were 3 ng/g and the lower limit of quantitation (LLOQ) was 30 ng/g.

Efficient synthesis of molecularly imprinted polymers with bio-recognition sites for the selective separation of bovine hemoglobin.

PubMed

Zhang, Zulei; Li, Lei

2018-06-01

We developed a facile approach to the construction of bio-recognition sites in silica nanoparticles for efficient separation of bovine hemoglobin based on amino-functionalized silica nanoparticles grafting by 3-aminopropyltriethoxylsilane providing hydrogen bonds with bovine hemoglobin through surface molecularly imprinting technology. The resulting amino-functionalized silica surface molecularly imprinted polymers were characterized using scanning electron microscope, transmission electronic microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Results showed that the as-synthesized imprinted polymers exhibited spherical morphology and favorable thermal stability. The binding adsorption experiments showed that the imprinted polymers can reach equilibrium within 1 h. The Langmuir isotherm and pseudo-second-order kinetic model fitted the adsorption data well. Meanwhile, the imprinted polymers possessed a maximum binding capacity up to 90.3 mg/g and highly selectivity for the recognition of bovine hemoglobin. Moreover, such high binding capacity and selectivity retained after eight cycles, indicating the good stability and reusability of the imprinted polymers. Finally, successful application in the selective recognition of bovine hemoglobin from a real bovine blood sample indicated that the imprinted polymers displayed great potentials in efficient purification and separation of target proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

PubMed

Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

2011-05-01

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Hybrid Phospholipid Bilayer Coatings for Separations of Cationic Proteins in Capillary Zone Electrophoresis

PubMed Central

Gallagher, Elyssia S.; Adem, Seid M.; Bright, Leonard K.; Calderon, Isen A. C.; Mansfield, Elisabeth; Aspinwall, Craig A.

2014-01-01

Protein separations in capillary zone electrophoresis (CZE) suffer from non-specific adsorption of analytes to the capillary surface. Semi-permanent phospholipid bilayers (PLBs) have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m−2, respectively, compared to 17 ± 1 mJ m−2 for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3 – 1.9 × 10−4 cm2 V−1s−1) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10−4 cm2 V−1s−1, 4.8 ± 0.4 × 10−4 cm2 V−1s−1, and 6.0 ± 0.2 × 10−4 cm2 V−1s−1, respectively), with increased stability compared to PLB coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6 %, n ≥ 6) with separation efficiencies as high as 200,000 plates m−1. PMID:24459085

A molecular modeling based method to predict elution behavior and binding patches of proteins in multimodal chromatography.

PubMed

Banerjee, Suvrajit; Parimal, Siddharth; Cramer, Steven M

2017-08-18

Multimodal (MM) chromatography provides a powerful means to enhance the selectivity of protein separations by taking advantage of multiple weak interactions that include electrostatic, hydrophobic and van der Waals interactions. In order to increase our understanding of such phenomena, a computationally efficient approach was developed that combines short molecular dynamics simulations and continuum solvent based coarse-grained free energy calculations in order to study the binding of proteins to Self Assembled Monolayers (SAM) presenting MM ligands. Using this method, the free energies of protein-MM SAM binding over a range of incident orientations of the protein can be determined. The resulting free energies were then examined to identify the more "strongly bound" orientations of different proteins with two multimodal surfaces. The overall free energy of protein-MM surface binding was then determined and correlated to retention factors from isocratic chromatography. This correlation, combined with analytical expressions from the literature, was then employed to predict protein gradient elution salt concentrations as well as selectivity reversals with different MM resin systems. Patches on protein surfaces that interacted strongly with MM surfaces were also identified by determining the frequency of heavy atom contacts with the atoms of the MM SAMs. A comparison of these patches to Electrostatic Potential and hydrophobicity maps indicated that while all of these patches contained significant positive charge, only the highest frequency sites also possessed hydrophobicity. The ability to identify key binding patches on proteins may have significant impact on process development for the separation of bioproduct related impurities. Copyright © 2017 Elsevier B.V. All rights reserved.

Phylogenetic analysis of the light-harvesting system in Chromera velia.

PubMed

Pan, Hao; Slapeta, Jan; Carter, Dee; Chen, Min

2012-03-01

Chromera velia is a newly discovered photosynthetic eukaryotic alga that has functional chloroplasts closely related to the apicoplast of apicomplexan parasites. Recently, the chloroplast in C. velia was shown to be derived from the red algal lineage. Light-harvesting protein complexes (LHC), which are a group of proteins involved in photon capture and energy transfer in photosynthesis, are important for photosynthesis efficiency, photo-adaptation/accumulation and photo-protection. Although these proteins are encoded by genes located in the nucleus, LHC peptides migrate and function in the chloroplast, hence the LHC may have a different evolutionary history compared to chloroplast evolution. Here, we compare the phylogenetic relationship of the C. velia LHCs to LHCs from other photosynthetic organisms. Twenty-three LHC homologues retrieved from C. velia EST sequences were aligned according to their conserved regions. The C. velia LHCs are positioned in four separate groups on trees constructed by neighbour-joining, maximum likelihood and Bayesian methods. A major group of seventeen LHCs from C. velia formed a separate cluster that was closest to dinoflagellate LHC, and to LHC and fucoxanthin chlorophyll-binding proteins from diatoms. One C. velia LHC sequence grouped with LI1818/LI818-like proteins, which were recently identified as environmental stress-induced protein complexes. Only three LHC homologues from C. velia grouped with the LHCs from red algae.

Process boundaries of irreversible scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

PubMed

Brandenbusch, Christoph; Glonke, Sebastian; Collins, Jonathan; Hoffrogge, Raimund; Grunwald, Klaudia; Bühler, Bruno; Schmid, Andreas; Sadowski, Gabriele

2015-11-01

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis. © 2015 Wiley Periodicals, Inc.

Fabrication of diverse pH-sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples.

PubMed

Wang, Jiaojiao; Lan, Jingfeng; Li, Huihui; Liu, Xiaoyan; Zhang, Haixia

2017-01-01

In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH-sensitive functional mesoporous silica materials composed of 2-(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH-sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five-protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum albumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.

PubMed

Wang, Hongbin; Hu, Gaofei; Zhang, Yongqian; Yuan, Zheng; Zhao, Xuan; Zhu, Yong; Cai, De; Li, Yujuan; Xiao, Shengyuan; Deng, Yulin

2010-07-15

The post-digestion (18)O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion (18)O labeling, the optimal (18)O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1-2 M and pH 4.5, 98.4+/-1.9% for a standard protein mixture and 97.2+/-6.2% for a complex biological sample. For a 1:1 mixture analysis of the (16)O- and (18)O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05+/-0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion (18)O labeling method. 2010 Elsevier B.V. All rights reserved.

Exosome separation using microfluidic systems: size-based, immunoaffinity-based and dynamic methodologies.

PubMed

Yang, Fang; Liao, Xiangzhi; Tian, Yuan; Li, Guiying

2017-04-01

Exosomes, nanovesicles secreted by most types of cells, exist in virtually all bodily fluids. Their rich nucleic acid and protein content make them potentially valuable biomarkers for noninvasive molecular diagnostics. They also show promise, after further development, to serve as a drug delivery system. Unfortunately, existing exosome separation technologies, such as ultracentrifugation and methods incorporating magnetic beads, are time-consuming, laborious and separate only exosomes of low purity. Thus, a more effective separation method is highly desirable. Microfluidic platforms are ideal tools for exosome separation, since they enable fast, cost-efficient, portable and precise processing of nanoparticles and small volumes of liquid samples. Recently, several microfluidic-based exosome separation technologies have been studied. In this article, the advantages of the most recent technologies, as well as their limitations, challenges and potential uses in novel microfluidic exosome separation and collection applications is reviewed. This review outlines the uses of new powerful microfluidic exosome detection tools for biologists and clinicians, as well as exosome separation tools for microfluidic engineers. Current challenges of exosome separation methodologies are also described, in order to highlight areas for future research and development. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ice-shell purification of ice-binding proteins.

PubMed

Marshall, Craig J; Basu, Koli; Davies, Peter L

2016-06-01

Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

PubMed

Vaganan, M Mayil; Sarumathi, S; Nandakumar, A; Ravi, I; Mustaffa, M M

2015-02-01

Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

Bio-inspired photo-electronic material based on photosynthetic proteins

NASA Astrophysics Data System (ADS)

Lebedev, Nikolai; Trammell, Scott A.; Tsoi, Stanislav; Spano, Anthony; Kim, Jin Ho; Xu, Jimmy; Twigg, Mark E.; Schnur, Joel M.

2009-08-01

The construction of efficient light energy converting (photovoltaic and photo-electronic) devices is a current and great challenge in science and technology and one that will have important economic consequences. Several innovative nanoelectronic materials were proposed to achieve this goal, semiconductor quantum dots, metallic nanowires and carbon nanotubes (CNT) are among them. As a charge separating unit for light energy conversion, we propose the utilization of the most advanced photoelectronic material developed by nature, photosynthetic reaction center proteins. As a first step in this direction, we constructed a novel bioinorganic nanophotoelectronic material with photoactive photosynthetic reaction center (RC) proteins encapsulated inside a multiwall CNT arrayed electrode. The material consists of photosynthetic RC-cytochrome complexes acting as charge separating units bound to the inner walls of a CNT electrode and ubiquinone-10 (Q2) serving as a soluble electron-transfer mediator to the counter electrode. The proteins were immobilized inside carbon nanotubes by a Ni(NTA)-alkane-pyrene linker, forming a self-assembled monolayer (SAM) on the surface of inner CNT walls and allowing for unidirectional protein orientation. The material demonstrates an enhanced photoinduced electron transfer rate and shows substantial improvement in photocurrent density compared to that obtained with the same proteins when immobilized on planar graphite (HOPG) electrode. The results suggest that protein encapsulation in precisely organized arrayed tubular electrode architecture can considerably improve the performance of photovoltaic, photoelectronic, or biofuel cell devices. They demonstrate the potential for substantial advantages of precisely organized nano electrode tubular arrayed architecture for variety biotechnological applications.

A Decaheme Cytochrome as a Molecular Electron Conduit in Dye-Sensitized Photoanodes

PubMed Central

Hwang, Ee Taek; Sheikh, Khizar; Orchard, Katherine L; Hojo, Daisuke; Radu, Valentin; Lee, Chong-Yong; Ainsworth, Emma; Lockwood, Colin; Gross, Manuela A; Adschiri, Tadafumi; Reisner, Erwin; Butt, Julea N; Jeuken, Lars J C

2015-01-01

In nature, charge recombination in light-harvesting reaction centers is minimized by efficient charge separation. Here, it is aimed to mimic this by coupling dye-sensitized TiO2 nanocrystals to a decaheme protein, MtrC from Shewanella oneidensis MR-1, where the 10 hemes of MtrC form a ≈7-nm-long molecular wire between the TiO2 and the underlying electrode. The system is assembled by forming a densely packed MtrC film on an ultra-flat gold electrode, followed by the adsorption of approximately 7 nm TiO2 nanocrystals that are modified with a phosphonated bipyridine Ru(II) dye (RuP). The step-by-step construction of the MtrC/TiO2 system is monitored with (photo)electrochemistry, quartz-crystal microbalance with dissipation (QCM-D), and atomic force microscopy (AFM). Photocurrents are dependent on the redox state of the MtrC, confirming that electrons are transferred from the TiO2 nanocrystals to the surface via the MtrC conduit. In other words, in these TiO2/MtrC hybrid photodiodes, MtrC traps the conduction-band electrons from TiO2 before transferring them to the electrode, creating a photobioelectrochemical system in which a redox protein is used to mimic the efficient charge separation found in biological photosystems. PMID:26180522

Extremely stable soluble high molecular mass multi-protein complex with DNase activity in human placental tissue.

PubMed

Burkova, Evgeniya E; Dmitrenok, Pavel S; Sedykh, Sergey E; Buneva, Valentina N; Soboleva, Svetlana E; Nevinsky, Georgy A

2014-01-01

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼ 1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4-14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography.

PubMed

Muck, A; Ramm, M; Hamburger, M

1999-09-10

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol-water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.

Adsorption and separation of proteins by collagen fiber adsorbent.

PubMed

Li, Juan; Liao, Xue-pin; Zhang, Qi-xian; Shi, Bi

2013-06-01

The separation of proteins is a key step in biomedical and pharmaceutical industries. In the present investigation, the collagen fiber adsorbent (CFA) was exploited as column packing material to separate proteins. Bovine serum albumin (BSA), bovine hemoglobin (Hb) and lysozyme (LYS) that have different isoelectric points (pIs) were selected as model proteins to investigate the separation ability of CFA to proteins. In batch adsorption, the adsorption behaviors of these proteins on CFA under different pHs and ionic strengths indicated that the electrostatic interaction plays a predominant role in the adsorption of proteins on CFA. CFA exhibited high adsorption capacity to Hb and LYS. In column separation, the proteins were completely separated by adjusting pH and ionic strength of the eluent. The increase of flow rate could reduce the separation time with no influence on the recovery of protein in the experimental range. The protein recovery was higher than 90% even when the CFA column was re-used for 4 times in separation of BSA and LYS, and the retention time of BSA or LYS was almost constant during the repeated applications. In addition, as a practical application, LYS was successfully separated from chicken egg white powder by CFA column. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomic analysis of processing by-products from canned and fresh tuna: identification of potentially functional food proteins.

PubMed

Sanmartín, Esther; Arboleya, Juan Carlos; Iloro, Ibon; Escuredo, Kepa; Elortza, Felix; Moreno, F Javier

2012-09-15

Proteomic approaches have been used to identify the main proteins present in processing by-products generated by the canning tuna-industry, as well as in by-products derived from filleting of skeletal red muscle of fresh tuna. Following fractionation by using an ammonium sulphate precipitation method, three proteins (tropomyosin, haemoglobin and the stress-shock protein ubiquitin) were identified in the highly heterogeneous and heat-treated material discarded by the canning-industry. Additionally, this fractionation method was successful to obtain tropomyosin of high purity from the heterogeneous starting material. By-products from skeletal red muscle of fresh tuna were efficiently fractionated to sarcoplasmic and myofibrillar fractions, prior to the identification based mainly on the combined searching of the peptide mass fingerprint (MALDI-TOF) and peptide fragment fingerprinting (MALDI LIFT-TOF/TOF) spectra of fifteen bands separated by 1D SDS-PAGE. Thus, the sarcoplasmic fraction contained myoglobin and several enzymes that are essential for efficient energy production, whereas the myofibrillar fraction had important contractile proteins, such as actin, tropomyosin, myosin or an isoform of the enzyme creatine kinase. Application of proteomic technologies has revealed new knowledge on the composition of important by-products from tuna species, enabling a better evaluation of their potential applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunit in Vivo.

PubMed

Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

2018-01-01

A leading objective in biology is to identify the complete set of activities that each gene performs in vivo In this study, we have asked whether a genetic approach can provide an efficient means of achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function ( sof - ) mutations in a gene. Toward this goal, we have subjected the Saccharomyces cerevisiae EST1 gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of sof - mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an est1 -∆ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of sof - mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell. Copyright © 2018 by the Genetics Society of America.

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

PubMed Central

Klotzsch, Enrico; Smorodchenko, Alina; Löfler, Lukas; Moldzio, Rudolf; Parkinson, Elena; Schütz, Gerhard J.; Pohl, Elena E.

2015-01-01

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. PMID:25535394

Separation of milk fat globules via microfiltration: Effect of diafiltration media and opportunities for stream valorization.

PubMed

Jukkola, A; Partanen, R; Rojas, O J; Heino, A

2016-11-01

Milk fat globule membranes (MFGM) sourced in buttermilk have gained recent interest given their nutritional value and functional properties. However, production of isolated MFGM has been challenging given their size similarity with casein micelles, which limits attempts toward fractionation by size exclusion techniques. Therefore, the hypothesis underpinning this study is that the removal of proteins from cream before butter-making facilitates MFGM isolation. As such, milk fat globules were separated from raw whole milk via microfiltration (1.4-µm pore diameter and 0.005-m 2 filtration surface area) by using 3 diafiltration media; namely, skim milk ultrafiltration permeate, saline, and water. Their effects on the stability of the milk fat globules and protein permeation was elucidated. Whereas a substantial reduction in protein concentration was achieved with all diafiltration media (~90% reduction), water and saline produced negligible membrane fouling with better filtration performance. Moreover, diafiltration with skim milk ultrafiltration permeate exhibited reduced permeate flux. Colloidal stability of the resultant milk decreased with all diafiltration solutions due to changing composition and reduced apparent viscosity. Overall, microfiltration was found to be an efficient method for separation of milk fat globules from whole milk, leading to increased MFGM fragment concentration in buttermilk dry matter, thus making it more suitable for industrial utilization. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

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

Qu, Yi; Feng, Ju; Deng, Shuang

2014-11-19

Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis ofmore » the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.« less

Temperature-Switchable Agglomeration of Magnetic Particles Designed for Continuous Separation Processes in Biotechnology.

PubMed

Paulus, Anja S; Heinzler, Raphael; Ooi, Huey Wen; Franzreb, Matthias

2015-07-08

The purpose of this work was the synthesis and characterization of thermally switchable magnetic particles for use in biotechnological applications such as protein purification and enzymatic conversions. Reversible addition-fragmentation chain-transfer polymerization was employed to synthesize poly(N-isopropylacrylamide) brushes via a "graft-from" approach on the surface of magnetic microparticles. The resulting particles were characterized by infrared spectroscopy and thermogravimetric analysis and their temperature-dependent agglomeration behavior was assessed. The influence of several factors on particle agglomeration (pH, temperature, salt type, and particle concentration) was evaluated. The results showed that a low pH value (pH 3-4), a kosmotropic salt (ammonium sulfate), and a high particle concentration (4 g/L) resulted in improved agglomeration at elevated temperature (40 °C). Recycling of particles and reversibility of the temperature-switchable agglomeration were successfully demonstrated for ten heating-cooling cycles. Additionally, enhanced magnetic separation was observed for the modified particles. Ionic monomers were integrated into the polymer chain to create end-group functionalized particles as well as two- and three-block copolymer particles for protein binding. The adsorption of lactoferrin, bovine serum albumin, and lysozyme to these ion exchange particles was evaluated and showed a binding capacity of up to 135 mg/g. The dual-responsive particles combined magnetic and thermoresponsive properties for switchable agglomeration, easy separability, and efficient protein adsorption.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

PubMed

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

2014-07-04

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

Mining the human plasma proteome with three-dimensional strategies by high-resolution Quadrupole Orbitrap Mass Spectrometry.

PubMed

Zhao, Yan; Chang, Cheng; Qin, Peibin; Cao, Qichen; Tian, Fang; Jiang, Jing; Li, Xianyu; Yu, Wenfeng; Zhu, Yunping; He, Fuchu; Ying, Wantao; Qian, Xiaohong

2016-01-21

Human plasma is a readily available clinical sample that reflects the status of the body in normal physiological and disease states. Although the wide dynamic range and immense complexity of plasma proteins are obstacles, comprehensive proteomic analysis of human plasma is necessary for biomarker discovery and further verification. Various methods such as immunodepletion, protein equalization and hyper fractionation have been applied to reduce the influence of high-abundance proteins (HAPs) and to reduce the high level of complexity. However, the depth at which the human plasma proteome has been explored in a relatively short time frame has been limited, which impedes the transfer of proteomic techniques to clinical research. Development of an optimal strategy is expected to improve the efficiency of human plasma proteome profiling. Here, five three-dimensional strategies combining HAP depletion (the 1st dimension) and protein fractionation (the 2nd dimension), followed by LC-MS/MS analysis (the 3rd dimension) were developed and compared for human plasma proteome profiling. Pros and cons of the five strategies are discussed for two issues: HAP depletion and complexity reduction. Strategies A and B used proteome equalization and tandem Seppro IgY14 immunodepletion, respectively, as the first dimension. Proteome equalization (strategy A) was biased toward the enrichment of basic and low-molecular weight proteins and had limited ability to enrich low-abundance proteins. By tandem removal of HAPs (strategy B), the efficiency of HAP depletion was significantly increased, whereas more off-target proteins were subtracted simultaneously. In the comparison of complexity reduction, strategy D involved a deglycosylation step before high-pH RPLC separation. However, the increase in sequence coverage did not increase the protein number as expected. Strategy E introduced SDS-PAGE separation of proteins, and the results showed oversampling of HAPs and identification of fewer proteins. Strategy C combined single Seppro IgY14 immunodepletion, high-pH RPLC fractionation and LC-MS/MS analysis. It generated the largest dataset, containing 1544 plasma protein groups and 258 newly identified proteins in a 30-h-machine-time analysis, making it the optimum three-dimensional strategy in our study. Further analysis of the integrated data from the five strategies showed identical distribution patterns in terms of sequence features and GO functional analysis with the 1929-plasma-protein dataset, further supporting the reliability of our plasma protein identifications. The characterization of 20 cytokines in the concentration range from sub-nanograms/milliliter to micrograms/milliliter demonstrated the sensitivity of the current strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Demonstration of a strategy for product purification by high-gradient magnetic fishing: recovery of superoxide dismutase from unconditioned whey.

PubMed

Meyer, Andrea; Hansen, Dennis B; Gomes, Cláudia S G; Hobley, Timothy J; Thomas, Owen R T; Franzreb, Matthias

2005-01-01

A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high-gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.15-0.6 mg L(-1)) in whey. The first part of the process design consisted of ligand screening in which metal chelate supports charged with copper(II) ions were found to be the most suitable. The second stage involved systematic and sequential optimization of conditions for the following steps: product adsorption, support washing, and product elution. Next, the capacity of a novel high-gradient magnetic separator (designed for biotechnological applications) for trapping and holding magnetic supports was determined. Finally, all of the above elements were assembled to deliver a HGMF process for the isolation of SOD from crude sweet whey, which consisted of (i) binding SOD using Cu2+ -charged magnetic metal chelator particles in a batch reactor with whey; (ii) recovery of the "SOD-loaded" supports by high-gradient magnetic separation (HGMS); (iii) washing out loosely bound and entrained proteins and solids; (iv) elution of the target protein; and (v) recovery of the eluted supports from the HGMF rig. Efficient recovery of SOD was demonstrated at approximately 50-fold increased scale (cf magnetic rack studies) in three separate HGMF experiments, and in the best of these (run 3) an SOD yield of >85% and purification factor of approximately 21 were obtained.

Rapid calibrated high-resolution hyperspectral imaging using tunable laser source

NASA Astrophysics Data System (ADS)

Nguyen, Lam K.; Margalith, Eli

2009-05-01

We present a novel hyperspectral imaging technique based on tunable laser technology. By replacing the broadband source and tunable filters of a typical NIR imaging instrument, several advantages are realized, including: high spectral resolution, highly variable field-of-views, fast scan-rates, high signal-to-noise ratio, and the ability to use optical fiber for efficient and flexible sample illumination. With this technique, high-resolution, calibrated hyperspectral images over the NIR range can be acquired in seconds. The performance of system features will be demonstrated on two example applications: detecting melamine contamination in wheat gluten and separating bovine protein from wheat protein in cattle feed.

The role of algal organic matter in the separation of algae and cyanobacteria using the novel "Posi" - Dissolved air flotation process.

PubMed

Hanumanth Rao, Narasinga Rao; Yap, Russell; Whittaker, Michael; Stuetz, Richard M; Jefferson, Bruce; Peirson, William L; Granville, Anthony M; Henderson, Rita K

2018-03-01

Algae and cyanobacteria frequently require separation from liquid media in both water treatment and algae culturing for biotechnology applications. The effectiveness of cell separation using a novel dissolved air flotation process that incorporates positively charged bubbles (PosiDAF) has recently been of interest but has been shown to be dependent on the algae or cyanobacteria species tested. Previously, it was hypothesised that algal organic matter (AOM) could be impacting the separation efficiency. Hence, this study investigates the influence of AOM on cell separation using PosiDAF, in which bubbles are modified using a commercially available cationic polyelectrolyte poly(N, N-diallyl-N,N-dimethylammonium chloride) (PDADMAC). The separation of Chlorella vulgaris CS-42/7, Mychonastes homosphaera CS-556/01 and two strains of Microcystis aeruginosa (CS-564/01 and CS-555/1), all of which have similar cell morphology but different AOM character, was investigated. By testing the cell separation in the presence and absence of AOM, it was determined that AOM enhanced cell separation for all the strains but to different extents depending on the quantity and composition of carbohydrates and proteins in the AOM. By extracting AOM from the strain for which optimal separation was observed and adding it to the others, cell separation improved from <55% to >90%. This was attributed to elevated levels of acidic carbohydrates as well as glycoprotein-carbohydrate conjugations, which in turn were related to the nature and quantity of proteins and carbohydrates present in the AOM. Therefore, it was concluded that process optimisation requires an in-depth understanding of the AOM and its components. If culturing algae for biotechnology applications, this indicates that strain selection is not only important with respect to high value product content, but also for cell separation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

PubMed Central

MacAlpine, Heather K.; Lubelsky, Yoav; Hartemink, Alexander J.

2015-01-01

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2–7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we “footprinted” nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2–7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). PMID:25593310

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

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

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE PAGES

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; ...

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

PubMed Central

Rashid, Fahad; Harris, Paul D; Zaher, Manal S; Sobhy, Mohamed A; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir M

2017-01-01

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.001 PMID:28230529

Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: noncovalent labeling and nonequilibrium measurement of association constants.

PubMed

Sloat, Amy L; Roper, Michael G; Lin, Xiuli; Ferrance, Jerome P; Landers, James P; Colyer, Christa L

2008-08-01

In response to a growing interest in the use of smaller, faster microchip (mu-chip) methods for the separation of proteins, advancements are proposed that employ the asymmetric squarylium dye Red-1c as a noncovalent label in mu-chip CE separations. This work compares on-column and precolumn labeling methods for the proteins BSA, beta-lactoglobulin B (beta-LB), and alpha-lactalbumin (alpha-LA). Nonequilibrium CE of equilibrium mixtures (NECEEM) represents an efficient method to determine equilibrium parameters associated with the formation of intermolecular complexes, such as those formed between the dye and proteins in this work, and it allows for the use of weak affinity probes in protein quantitation. In particular, nonequilibrium methods employing both mu-chip and conventional CE systems were implemented to determine association constants governing the formation of noncovalent complexes of the red luminescent squarylium dye Red-1c with BSA and beta-LB. By our mu-chip NECEEM method, the association constants K(assoc) for beta-LB and BSA complexes with Red-1c were found to be 3.53 x 10(3) and 1.65 x 10(5) M(-1), respectively, whereas association constants found by our conventional CE-LIF NECEEM method for these same protein-dye systems were some ten times higher. Despite discrepancies between the two methods, both confirmed the preferential interaction of Red-1c with BSA. In addition, the effect of protein concentration on measured association constant was assessed by conventional CE methods. Although a small decrease in K(assoc) was observed with the increase in protein concentration, our studies indicate that absolute protein concentration may affect the equilibrium determination less than the relative concentration of protein-to-dye.

Recyclability of PET/WPI/PE Multilayer Films by Removal of Whey Protein Isolate-Based Coatings with Enzymatic Detergents.

PubMed

Cinelli, Patrizia; Schmid, Markus; Bugnicourt, Elodie; Coltelli, Maria Beatrice; Lazzeri, Andrea

2016-06-14

Multilayer plastic films provide a range of properties, which cannot be obtained from monolayer films but, at present, their recyclability is an open issue and should be improved. Research to date has shown the possibility of using whey protein as a layer material with the property of acting as an excellent barrier against oxygen and moisture, replacing petrochemical non-recyclable materials. The innovative approach of the present research was to achieve the recyclability of the substrate films by separating them, with a simple process compatible with industrial procedures, in order to promote recycling processes leading to obtain high value products that will beneficially impact the packaging and food industries. Hence, polyethyleneterephthalate (PET)/polyethylene (PE) multi-layer film was prepared based on PET coated with a whey protein layer, and then the previous structure was laminated with PE. Whey proteins, constituting the coating, can be degraded by enzymes so that the coating films can be washed off from the plastic substrate layer. Enzyme types, dosage, time, and temperature optima, which are compatible with procedures adopted in industrial waste recycling, were determined for a highly-efficient process. The washing of samples based on PET/whey and PET/whey/PE were efficient when performed with enzymatic detergent containing protease enzymes, as an alternative to conventional detergents used in recycling facilities. Different types of enzymatic detergents tested presented positive results in removing the protein layer from the PET substrate and from the PET/whey/PE multilayer films at room temperature. These results attested to the possibility of organizing the pre-treatment of the whey-based multilayer film by washing with different available commercial enzymatic detergents in order to separate PET and PE, thus allowing a better recycling of the two different polymers. Mechanical properties of the plastic substrate, such as stress at yield, stress and elongation at break, evaluated by tensile testing on films before and after cleaning, were are not significantly affected by washing with enzymatic detergents.

The role of energy losses in photosynthetic light harvesting

NASA Astrophysics Data System (ADS)

Krüger, T. P. J.; van Grondelle, R.

2017-07-01

Photosynthesis operates at the bottom of the food chain to convert the energy of light into carbohydrates at a remarkable global rate of about 130 TW. Nonetheless, the overall photosynthetic process has a conversion efficiency of a few percent at best, significantly less than bottom-up photovoltaic cells. The primary photosynthetic steps, consisting of light harvesting and charge separation, are often presented as having near-unity quantum efficiency but this holds only true under ideal conditions. In this review, we discuss the importance of energy loss mechanisms to establish robustness in photosynthetic light harvesting. Thermal energy dissipation of light-harvesting complexes (LHCs) in different environments is investigated and the relationships and contrasts between concentration quenching of high pigment concentrations, photoprotection (non-photochemical quenching), quenching due to protein aggregation, and fluorescence blinking are discussed. The role of charge-transfer states in light harvesting and energy dissipation is highlighted and the importance of controlled protein structural disorder to switch the light-harvesting antennae between effective light harvesters and efficient energy quenchers is underscored. The main LHC of plants, LHCII, is used as a prime example.

A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

PubMed Central

Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

2009-01-01

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes. PMID:19290621

Assembly of Photosynthetic Antenna Protein Complexes from Algae for Development of Nano-biodevice and Its Fuelization

DTIC Science & Technology

2013-05-20

More recently the structures of the LH2 complexes has revealed the nonameric and octameric arrangement of repeating units consisting of two...Compartimentalization of light -harvesting and charge separation. The antenna complexes( LH2 ,LH1-RC) efficiently realize various photosynthetic functions...using cofactors (BChl a and carotenoid) assembled into the apoproteins (LH1 and LH2 ). The light-harvesting mechanisms in these light-harvesting

Assembly of Photosynthetic Antenna Protein / Pigments Complexes from Algae and Plants for Development of Nanobiodevices

DTIC Science & Technology

2012-07-10

recently the structures of the LH2 complexes has revealed the nonameric and octameric arrangement of repeating units consisting of two apoproteins and...Compartimentalization of light -harvesting and charge separation. The antenna complexes( LH2 ,LH1-RC) efficiently realize various photosynthetic functions using...cofactors (BChl a and carotenoid) assembled into the apoproteins (LH1 and LH2 ). The light-harvesting mechanisms in these light-harvesting complexes have

High pH instability of quaternary ammonium surfactant coatings in capillary electrophoresis.

PubMed

Shulman, Lisa; Pei, Lei; Bahnasy, Mahmoud F; Lucy, Charles A

2017-06-12

The two-tailed cationic surfactant dioctadecyldimethyl ammonium bromide (DODAB) produces semi-permanent coatings that yield strongly reversed electroosmotic flow (EOF), for example -0.31 ± 0.01 cm 2 kV -1 s -1 at pH 3.5. Moreover, these coatings are easy to prepare, regenerable, cost effective, and yield high efficiency (520 000-900 000 plates per m) separations of cationic proteins over many runs under acidic (pH 3.5) conditions. Given the quaternary amine functionality of DODAB, we were surprised to observe that DODAB coatings become unstable at pH > 7. At pH 7.2, the EOF of a DODAB coated capillary drifted from reversed to cathodic over only 5 runs, and protein separations became severely compromised. By pH 12, no EOF reversal was observed. Electrophoretic and mass spectrometric studies demonstrate that the coating decomposition involves a surface conversion of the quaternary amine in DODAB to a variety of products, although the exact mechanism remains elusive. Regardless, the results herein demonstrate that semi-permanent coatings based on cationic two-tailed surfactants such as DODAB are limited to separations using acidic buffers.

Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives

PubMed Central

Edelmann, Mariola J.

2011-01-01

Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide. PMID:22174558

Dynamic multiprotein assemblies shape the spatial structure of cell signaling.

PubMed

Nussinov, Ruth; Jang, Hyunbum

2014-01-01

Cell signaling underlies critical cellular decisions. Coordination, efficiency as well as fail-safe mechanisms are key elements. How the cell ensures that these hallmarks are at play are important questions. Cell signaling is often viewed as taking place through discrete and cross-talking pathways; oftentimes these are modularized to emphasize distinct functions. While simple, convenient and clear, such models largely neglect the spatial structure of cell signaling; they also convey inter-modular (or inter-protein) spatial separation that may not exist. Here our thesis is that cell signaling is shaped by a network of multiprotein assemblies. While pre-organized, the assemblies and network are loose and dynamic. They contain transiently-associated multiprotein complexes which are often mediated by scaffolding proteins. They are also typically anchored in the membrane, and their continuum may span the cell. IQGAP1 scaffolding protein which binds proteins including Raf, calmodulin, Mek, Erk, actin, and tens more, with actin shaping B-cell (and likely other) membrane-anchored nanoclusters and allosterically polymerizing in dynamic cytoskeleton formation, and Raf anchoring in the membrane along with Ras, provides a striking example. The multivalent network of dynamic proteins and lipids, with specific interactions forming and breaking, can be viewed as endowing gel-like properties. Collectively, this reasons that efficient, productive and reliable cell signaling takes place primarily through transient, preorganized and cooperative protein-protein interactions spanning the cell rather than stochastic, diffusion-controlled processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effective water content reduction in sewage wastewater sludge using magnetic nanoparticles.

PubMed

Lakshmanan, Ramnath; Kuttuva Rajarao, Gunaratna

2014-02-01

The present work compares the use of three flocculants for sedimentation of sludge and sludge water content from sewage wastewater i.e. magnetic iron oxide nanoparticles (MION), ferrous sulfate (chemical) and Moringa crude extract (protein). Sludge water content, wet/dry weight, turbidity and color were performed for, time kinetics and large-scale experiment. A 30% reduction of the sludge water content was observed when the wastewater was treated with either protein or chemical coagulant. The separation of sludge from wastewater treated with MION was achieved in less than 5min using an external magnet, resulted in 95% reduction of sludge water content. Furthermore, MION formed denser flocs and more than 80% reduction of microbial content was observed in large volume experiments. The results revealed that MION is efficient in rapid separation of sludge with very low water content, and thus could be a suitable alternative for sludge sedimentation and dewatering in wastewater treatment processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry.

PubMed

Sorensen, Matthew; Harmes, David C; Stoll, Dwight R; Staples, Gregory O; Fekete, Szabolcs; Guillarme, Davy; Beck, Alain

2016-10-01

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

SnipViz: a compact and lightweight web site widget for display and dissemination of multiple versions of gene and protein sequences.

PubMed

Jaschob, Daniel; Davis, Trisha N; Riffle, Michael

2014-07-23

As high throughput sequencing continues to grow more commonplace, the need to disseminate the resulting data via web applications continues to grow. Particularly, there is a need to disseminate multiple versions of related gene and protein sequences simultaneously--whether they represent alleles present in a single species, variations of the same gene among different strains, or homologs among separate species. Often this is accomplished by displaying all versions of the sequence at once in a manner that is not intuitive or space-efficient and does not facilitate human understanding of the data. Web-based applications needing to disseminate multiple versions of sequences would benefit from a drop-in module designed to effectively disseminate these data. SnipViz is a client-side software tool designed to disseminate multiple versions of related gene and protein sequences on web sites. SnipViz has a space-efficient, interactive, and dynamic interface for navigating, analyzing and visualizing sequence data. It is written using standard World Wide Web technologies (HTML, Javascript, and CSS) and is compatible with most web browsers. SnipViz is designed as a modular client-side web component and may be incorporated into virtually any web site and be implemented without any programming. SnipViz is a drop-in client-side module for web sites designed to efficiently visualize and disseminate gene and protein sequences. SnipViz is open source and is freely available at https://github.com/yeastrc/snipviz.

Dirt feedlot residue experiments. Quarterly progress report, December 1977--March 1978

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

Turk, M.

1978-04-01

Performance of the mobile fermentation system is reported. It made use of aged pen residue at the nominal loading rate of 0.25 lbs. volatile solids/ft./sup 3//day with a 10-day retention time and a fermentation temperature of 57/sup 0/C. Results of an experimental cattle feeding trial utilizing the protein in the fermentor liquid effluent as a replacement for standard protein supplements were encouraging. The evaluation of the capture efficiency of the system centrifuge both with and without a chemical flocculant was completed. An experimental cattle feeding trial utilizing the protein fermentation product (PFP) harvested by the centrifuge as replacement for themore » standard protein supplementwas initiated. The characterization of the cattle residues found in various cattle pens, feedlots, and locations was continued. An investigation was initiated into methods of separating the organic content of the feedlot residue from the sand and grit content. (JGB)« less

Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips†

PubMed Central

Earhart, Christopher M.; Hughes, Casey E.; Gaster, Richard S.; Ooi, Chin Chun; Wilson, Robert J.; Zhou, Lisa Y.; Humke, Eric W.; Xu, Lingyun; Wong, Dawson J.; Willingham, Stephen B.; Schwartz, Erich J.; Weissman, Irving L.; Jeffrey, Stefanie S.; Neal, Joel W.; Rohatgi, Rajat; Wakelee, Heather A.; Wang, Shan X.

2014-01-01

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of “liquid biopsies” from cancer patients. PMID:23969419

Fabrication of high-capacity polyelectrolyte brush-grafted porous AAO-silica composite membrane via RAFT polymerization.

PubMed

Song, Cunfeng; Wang, Meijie; Liu, Xin; Wang, He; Chen, Xiaoling; Dai, Lizong

2017-09-01

Surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization has been utilized to fabricate high-capacity strong anion-exchange (AEX) membrane for the separation of protein. By means of RAFT polymerization, quaternized poly(3-(methacrylamidomethyl)-pyridine) brushes formed 3-dimensional nanolayers on the surface of porous anodic aluminum oxide (AAO)-silica composite membrane. The surface properties of the membranes were analyzed by SEM, water contact angle, ATR-FTIR, XPS and TGA. To investigate the adsorption performance, the new AEX membranes were applied to recover a model protein, ovalbumin (OVA). High adsorption capacities of 95.8mg/g membranes (static) and 65.3mg/g membranes (dynamic) were obtained at ambient temperature. In the further studies, up to 90% of the adsorbed OVA was efficiently eluted by using phosphate buffer-1M NaCl as elution medium. The successful separation of OVA with high purity from a mixture protein solution was also achieved by using the AEX membranes. The present study demonstrated that under mild reaction condition, RAFT polymerization can be used to fabricate ion-exchange membrane which has many remarkable features, such as high capacity and selectivity, easy elution and so on. Copyright © 2017. Published by Elsevier B.V.

Investigation of electrodialysis anti-fouling configuration for desalting and treating tannery unhairing wastewater: Feasibility of by-products recovery and water recycling.

PubMed

Tamersit, Sabrina; Bouhidel, Kamel-Eddine; Zidani, Zakaria

2018-02-01

The desalination and treatment of tannery unhairing wastewater by electrodialysis (ED) is investigated in this research in order to separate, concentrate, recover and reuse low molecular weight charged species (S 2- , HS - , OH - , Cl - , Ca 2+ , Na + and amino acids), and to separate proteins and recycle treated water. Therefore, a novel electrodialysis membrane configuration was proposed. This was based on a double anti-fouling membrane. The ED anion exchange membrane (AEM), which is very sensitive to organic fouling, was protected by an ultra filtration membrane impermeable to the negatively charged proteins that could not reach the AEM surface. The experimental results were quite promising, and in spite of only one desalination compartment ED cell; the demineralization efficiency was 56 ± 1.25% (5.5-2.4 mS/cm), with a sensitive removal of sulphide, calcium and chloride. The organic matter (protein, peptides…) was isolated in the dilute compartment. The most important result was the total absence of membrane fouling. The experimental results remarkably proved the initial hypothesis, and suggested promising solutions for industrial pollution, where the membrane processes have never been successful. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis

PubMed Central

Pugsley, Haley R.; Swearingen, Kristian E.; Dovichi, Norman J.

2009-01-01

A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%. PMID:19249052

Versatile antifouling polyethersulfone filtration membranes modified via surface grafting of zwitterionic polymers from a reactive amphiphilic copolymer additive.

PubMed

Zhao, Yi-Fan; Zhang, Pei-Bin; Sun, Jian; Liu, Cui-Jing; Yi, Zhuan; Zhu, Li-Ping; Xu, You-Yi

2015-06-15

Here we describe the development of versatile antifouling polyethersulfone (PES) filtration membranes modified via surface grafting of zwitterionic polymers from a reactive amphiphilic copolymer additive. Amphiphilic polyethersulfone-block-poly(2-hydroxyethyl methacrylate) (PES-b-PHEMA) was beforehand designed and used as the blending additive of PES membranes prepared by phase inversion technique. The surface enriched PHEMA blocks on membrane surface acted as an anchor to immobilize the initiating site. Poly(sulfobetaine methacrylate) (PSBMA) were subsequently grafted onto the PES blend membranes by surface-initiated atom transfer radical polymerization (SI-ATRP). The analysis of surface chemistry confirmed the successful grafting of zwitterionic PSBMA brushes on PES membrane surface. The resulted PES-g-PSBMA membranes were capable of separating proteins from protein solution and oil from oil/water emulsion efficiently. Furthermore, the modified membranes showed high hydrophilicity and strongly antifouling properties due to the incorporation of well-defined PSBMA layer. In addition, the PES-g-PSBMA membranes exhibited excellent blood compatibility and durability during the washing process. The developed antifouling PES membranes are versatile and can find their applications in protein filtration, blood purification and oil/water separation, etc. Copyright © 2015 Elsevier Inc. All rights reserved.

Domain Formation Induced by the Adsorption of Charged Proteins on Mixed Lipid Membranes

PubMed Central

Mbamala, Emmanuel C.; Ben-Shaul, Avinoam; May, Sylvio

2005-01-01

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation. PMID:15626713

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network.

PubMed

Li, Yan; Buch, Jesse S; Rosenberger, Frederick; DeVoe, Don L; Lee, Cheng S

2004-02-01

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

PubMed Central

Falahati, Hanieh; Wieschaus, Eric

2017-01-01

Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision. PMID:28115706

Green and Sustainable Separation of Natural Products from Agro-Industrial Waste: Challenges, Potentialities, and Perspectives on Emerging Approaches.

PubMed

Zuin, Vânia G; Ramin, Luize Z

2018-01-17

New generations of biorefinery combine innovative biomass waste resources from different origins, chemical extraction and/or synthesis of biomaterials, biofuels, and bioenergy via green and sustainable processes. From the very beginning, identifying and evaluating all potentially high value-added chemicals that could be removed from available renewable feedstocks requires robust, efficient, selective, reproducible, and benign analytical approaches. With this in mind, green and sustainable separation of natural products from agro-industrial waste is clearly attractive considering both socio-environmental and economic aspects. In this paper, the concepts of green and sustainable separation of natural products will be discussed, highlighting the main studies conducted on this topic over the last 10 years. The principal analytical techniques (such as solvent, microwave, ultrasound, and supercritical treatments), by-products (e.g., citrus, coffee, corn, and sugarcane waste) and target compounds (polyphenols, proteins, essential oils, etc.) will be presented, including the emerging green and sustainable separation approaches towards bioeconomy and circular economy contexts.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

PubMed

Churion, Kelly A; Rogers, Robert E; Bayless, Kayla J; Bondos, Sarah E

2016-12-01

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation. Copyright © 2016 Elsevier Inc. All rights reserved.

HPLC-Chip/MS Technology in Proteomic Profiling

NASA Astrophysics Data System (ADS)

Vollmer, Martin; van de Goor, Tom

HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.

Binding of cadmium to Strychnos potatorum seed proteins in aqueous solution: adsorption kinetics and relevance to water purification.

PubMed

Saif, Mohammad Mansour Saleh; Kumar, N Siva; Prasad, M N V

2012-06-01

Strychnos potatorum seeds (cleaning nuts or nirmali) are extensively used by remote village tribals in the state of Andhra Pradesh, India for clarification of turbid and metal contaminated water. In the present study the ability of seed proteins to bind aqueous cadmium has been investigated. Biochemical characterization of the seed powder revealed the presence of coagulant proteins. These proteins were isolated from the soluble extracts of the seeds by ammonium sulfate fractionation. The (30-70%) fraction containing the bulk of proteins were separated by gel filtration into two peaks A and B. The (30-70%) ammonium sulfate precipitated proteins, as well as those from Peak A and B were separately immobilized to affigel-10. The Cd(II) biosorption efficiency by these proteins have been investigated. Different experiments have been conducted (i) over a range of pH (2.0-7.0), (ii) contact time (5-600 min), (iii) temperatures (4-40°C) and (iv) metal ion concentrations (80-110 mg L(-1)). The results showed that the optimum conditions for Cd(II) adsorption are almost same for the three proteins used in the study. Cd(II) removal is pH dependent and the maximum removal was at pH 5.0 in a time span of 360 min. The equilibrium data fit into Langmuir isotherm than Freundlich model. The correlation coefficient for the pseudo second order is high (~0.996-1.00) where as the correlation coefficient of the pseudo first order model is too low so the adsorption is better described by pseudo second order model. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

Recovery of protein hydrolysate and chitosan from black tiger shrimp (Penaeus monodon) heads: approaching a zero waste process.

PubMed

Phuong, Pham Thi Dan; Minh, Nguyen Cong; Cuong, Hoang Ngoc; Van Minh, Nguyen; Han, Nguyen The; Van Hoa, Nguyen; Yen, Ha Thi Hai; Trung, Trang Si

2017-06-01

Shrimp heads are considered as a potential source for the recovery of many valuable components such as chitin, protein and carotenoids. In the present study, both protein hydrolysate and chitin/chitosan were recovered using combination of physical, biological and chemical treatments. Shrimp heads were separated from liquid phase by a facile and efficient physical pretreatment. The liquid fraction was then hydrolyzed using formic acid and vacuum concentrated to obtain an astaxanthin-rich protein hydrolysate. The solid fraction was used to produce chitin by deproteination and demineralization. The hydrolysate consisted of a high astaxanthin (192 ppm) and protein (26.3 wt%) content. Chitosan prepared from chitin showed a high degree of deacetylation (85.4%) with low protein (0.25 wt%) and mineral (0.22 wt%) contents. The relative crystalline structure of the chitin and chitosan were 54.7 and 10.4%, respectively. The deacetylation of chitin was confirmed by Fourier Transform Infrared Spectroscopy. The present procedure approached to produce maximum valuable components including astaxanthin-rich protein hydrolysate and chitin/chitosan from shrimp head waste.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

PubMed

Huang, Sheng Yu; Chen, Sung Fang; Chen, Chun Hao; Huang, Hsuan Wei; Wu, Wen Guey; Sung, Wang Chou

2014-09-02

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Processing scale-up of sicklepod (Senna obtusifolia L.) seed.

PubMed

Harry-O'Kuru, Rogers E; Mohamed, Abdellatif

2009-04-08

Sicklepod (Senna obtusifolia L.) is an invasive weed species especially of soybean and other field crops in the southeastern United States. The seeds contain a small amount (5-7%) of a highly colored fat as well as various phenolics, proteins, and galactomannans. The color of sicklepod seed oil is such that the presence of a small amount of the weed seed in a soybean crush lowers the quality of the soybean oil. Sicklepod is very prolific, and even volunteer stands yield >1000 lb of seed per acre, and prudence calls for tapping the potential of this weed as an alternative economic crop in the affected region. Pursuant to this, we have shown in laboratory-scale work the feasibility of separating the components of sicklepod seed. However, at kilogram and higher processing quantities, difficulties arise leading to modification of the earlier approach in order to efficiently separate components of the defatted seed meal. In a version for cleanly separating the proteins, the defatted meal was extracted with 0.5 M NaCl solution to remove globular proteins. Prolamins were extracted from the pellet left after salt extraction using 80% ethanol, and glutelins were then obtained in 0.1 N alkali from the residual solids left from ethanol treatment. In a pilot-scale version for water-soluble polysaccharides, the defatted meal was stirred with deionized water (DI) and centrifuged. The pooled centrifugates were heated to 92 degrees C (20-25 min), filtered, cooled to room temperature, and passed through a column of Amberlite XAD-4 to separate the polysaccharides from the anthraquinones. Senna obtusifolia L. is a one-stop-shop of a seed (from food components to medicinals).

Quantifying condition-dependent intracellular protein levels enables high-precision fitness estimates.

PubMed

Geiler-Samerotte, Kerry A; Hashimoto, Tatsunori; Dion, Michael F; Budnik, Bogdan A; Airoldi, Edoardo M; Drummond, D Allan

2013-01-01

Countless studies monitor the growth rate of microbial populations as a measure of fitness. However, an enormous gap separates growth-rate differences measurable in the laboratory from those that natural selection can distinguish efficiently. Taking advantage of the recent discovery that transcript and protein levels in budding yeast closely track growth rate, we explore the possibility that growth rate can be more sensitively inferred by monitoring the proteomic response to growth, rather than growth itself. We find a set of proteins whose levels, in aggregate, enable prediction of growth rate to a higher precision than direct measurements. However, we find little overlap between these proteins and those that closely track growth rate in other studies. These results suggest that, in yeast, the pathways that set the pace of cell division can differ depending on the growth-altering stimulus. Still, with proper validation, protein measurements can provide high-precision growth estimates that allow extension of phenotypic growth-based assays closer to the limits of evolutionary selection.

Preservation of protein expression systems at elevated temperatures for portable therapeutic production

PubMed Central

Bessling, Seneca; Thielen, Peter; Zhang, Sherry; Wolfe, Joshua

2017-01-01

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability. PMID:28446704

Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

PubMed

Valeja, Santosh G; Xiu, Lichen; Gregorich, Zachery R; Guner, Huseyin; Jin, Song; Ge, Ying

2015-01-01

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica.

PubMed

Lee, Soo Youn; Lee, Jiho; Lee, Hye Sun; Chang, Jeong Ho

2014-03-15

We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step. © 2013 Elsevier B.V. All rights reserved.

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

PubMed

Huang, Wanfeng; Chang, Chun-Li; Brault, Norman D; Gur, Onur; Wang, Zhe; Jalal, Shadia I; Low, Philip S; Ratliff, Timothy L; Pili, Roberto; Savran, Cagri A

2017-01-31

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.

Strategies for the enrichment and identification of basic proteins in proteome projects.

PubMed

Bae, Soo-Han; Harris, Andrew G; Hains, Peter G; Chen, Hong; Garfin, David E; Hazell, Stuart L; Paik, Young-Ki; Walsh, Bradley J; Cordwell, Stuart J

2003-05-01

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors.

PubMed

Zhang, Peng; Gao, Mingxia; Zhu, Shaochun; Lei, Jie; Zhang, Xiangmin

2011-11-25

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification. Copyright © 2011 Elsevier B.V. All rights reserved.

Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

PubMed

Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

2014-05-01

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Safeguards of Neurotransmission: Endocytic Adaptors as Regulators of Synaptic Vesicle Composition and Function

PubMed Central

Kaempf, Natalie; Maritzen, Tanja

2017-01-01

Communication between neurons relies on neurotransmitters which are released from synaptic vesicles (SVs) upon Ca2+ stimuli. To efficiently load neurotransmitters, sense the rise in intracellular Ca2+ and fuse with the presynaptic membrane, SVs need to be equipped with a stringently controlled set of transmembrane proteins. In fact, changes in SV protein composition quickly compromise neurotransmission and most prominently give rise to epileptic seizures. During exocytosis SVs fully collapse into the presynaptic membrane and consequently have to be replenished to sustain neurotransmission. Therefore, surface-stranded SV proteins have to be efficiently retrieved post-fusion to be used for the generation of a new set of fully functional SVs, a process in which dedicated endocytic sorting adaptors play a crucial role. The question of how the precise reformation of SVs is achieved is intimately linked to how SV membranes are retrieved. For a long time both processes were believed to be two sides of the same coin since Clathrin-mediated endocytosis (CME), the proposed predominant SV recycling mode, will jointly retrieve SV membranes and proteins. However, with the recent proposal of Clathrin-independent SV recycling pathways SV membrane retrieval and SV reformation turn into separable events. This review highlights the progress made in unraveling the molecular mechanisms mediating the high-fidelity retrieval of SV proteins and discusses how the gathered knowledge about SV protein recycling fits in with the new notions of SV membrane endocytosis. PMID:29085282

Protein Separation by Capillary Gel Electrophoresis: A Review

PubMed Central

Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

2011-01-01

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

Selective adsorption of bovine hemoglobin on functional TiO2 nano-adsorbents: surface physic-chemical properties determined adsorption activity

NASA Astrophysics Data System (ADS)

Guo, Shiguang; Zhang, Jianghua; Shao, Mingxue; Zhang, Xia; Liu, Yufeng; Xu, Junli; Meng, Hao; Han, Yide

2015-04-01

Surface functionalized nanoparticles are efficient adsorbents which have shown good potential for protein separation. In this work, we chose two different types of organic molecules, oleic acid (OA) and 3-glycidoxypropyltrimethoxy silane (GPTMS), to functionalize the surface of TiO2 nanoparticles, and we studied the effects of this modification on their surface physicochemical properties in correlation with their selective adsorption of proteins. The results showed that the surface zeta potential and the surface water wettability of the modified TiO2 were significantly changed in comparison with the original TiO2 nanoparticles. The adsorption activities of bovine hemoglobin (BHb) and bovine serum albumin (BSA) on these functionalized TiO2 samples were investigated under different conditions, including pH values, contact time, ion strength, and initial protein concentration. In comparison with the non-specific adsorption of original TiO2, however, both the OA-TiO2 and GPTMS-TiO2 exhibited increased BHb adsorption and decreased BSA adsorption at the same time. Using a binary protein mixture as the adsorption object, a higher separation factor (SF) was obtained for OA-TiO2 under optimum conditions. The different adsorption activities of BHb and BSA on the modified TiO2 were correlated with different interactions at the protein/solid interface, and the chemical force as well as the electrostatic force played an important role in the selective adsorption process.

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging*

PubMed Central

Ramallo Guevara, Carina; Philipp, Oliver; Hamann, Andrea; Werner, Alexandra; Osiewacz, Heinz D.; Rexroth, Sascha; Rögner, Matthias; Poetsch, Ansgar

2016-01-01

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. PMID:26884511

Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

PubMed Central

Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

2007-01-01

Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

Detection of Singlet Oxygen Formation inside Photoactive Biohybrid Composite Material.

PubMed

Hajdu, Kata; Ur Rehman, Ateeq; Vass, Imre; Nagy, László

2017-12-26

Photosynthetic reaction center proteins (RCs) are the most efficient light energy converter systems in nature. The first steps of the primary charge separation in photosynthesis take place in these proteins. Due to their unique properties, combining RCs with nano-structures promising applications can be predicted in optoelectronic systems. In the present work RCs purified from Rhodobacter sphaeroides purple bacteria were immobilized on multiwalled carbon nanotubes (CNTs). Carboxyl-and amine-functionalised CNTs were used, so different binding procedures, physical sorption and chemical sorption as well, could be applied as immobilization techniques. Light-induced singlet oxygen production was measured in the prepared photoactive biocomposites in water-based suspension by histidine mediated chemical trapping. Carbon nanotubes were applied under different conditions in order to understand their role in the equilibration of singlet oxygen concentration in the suspension. CNTs acted as effective quenchers of ¹O₂ either by physical (resonance) energy transfer or by chemical (oxidation) reaction and their efficiency showed dependence on the diffusion distance of ¹O₂.

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

Fabrication of universal serial bus flash disk type microfluidic chip electrophoresis and application for protein analysis under ultra low voltage

PubMed Central

Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei

2016-01-01

A simple and effective universal serial bus (USB) flash disk type microfluidic chip electrophoresis (MCE) was developed by using poly(dimethylsiloxane) based soft lithography and dry film based printed circuit board etching techniques in this paper. The MCE had a microchannel diameter of 375 μm and an effective length of 25 mm. Equipped with a conventional online electrochemical detector, the device enabled effectively separation of bovine serum albumin, lysozyme, and cytochrome c in 80 s under the ultra low voltage from a computer USB interface. Compared with traditional capillary electrophoresis, the USB flash disk type MCE is not only portable and inexpensive but also fast with high separation efficiency. PMID:27042249

Drug Target Protein-Protein Interaction Networks: A Systematic Perspective

PubMed Central

2017-01-01

The identification and validation of drug targets are crucial in biomedical research and many studies have been conducted on analyzing drug target features for getting a better understanding on principles of their mechanisms. But most of them are based on either strong biological hypotheses or the chemical and physical properties of those targets separately. In this paper, we investigated three main ways to understand the functional biomolecules based on the topological features of drug targets. There are no significant differences between targets and common proteins in the protein-protein interactions network, indicating the drug targets are neither hub proteins which are dominant nor the bridge proteins. According to some special topological structures of the drug targets, there are significant differences between known targets and other proteins. Furthermore, the drug targets mainly belong to three typical communities based on their modularity. These topological features are helpful to understand how the drug targets work in the PPI network. Particularly, it is an alternative way to predict potential targets or extract nontargets to test a new drug target efficiently and economically. By this way, a drug target's homologue set containing 102 potential target proteins is predicted in the paper. PMID:28691014

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography.

PubMed

Gu, Dongyu; Lazo-Portugal, Rodrigo; Fang, Chen; Wang, Zhantong; Ma, Ying; Knight, Martha; Ito, Yoichiro

2018-06-15

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A 565 /A 280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ) 6 γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source. Copyright © 2018 Elsevier B.V. All rights reserved.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

PubMed

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-07-05

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins.

PubMed

Gai, Qingqing; Qu, Feng; Zhang, Tao; Zhang, Yukui

2011-07-15

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation. Copyright © 2011 Elsevier B.V. All rights reserved.

Separation of porcine parvovirus from bovine serum albumin using PEG-salt aqueous two-phase system.

PubMed

Vijayaragavan, K Saagar; Zahid, Amna; Young, Jonathan W; Heldt, Caryn L

2014-09-15

Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80-85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG-citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation. Copyright © 2014 Elsevier B.V. All rights reserved.

The effects of RPM and recycle on separation efficiency in a clinical blood cell centrifuge.

PubMed

Drumheller, P D; Van Wie, B J; Petersen, J N; Oxford, R J; Schneider, G W

1987-11-01

A COBE blood cell centrifuge, model 2997 with a single stage channel, was modified to allow computer controlled sampling, and to allow recycle of red blood cells (RBCs) and plasma streams using bovine whole blood. The effects of recycle of the packed RBC and plasma product streams, and of the centrifuge RPM on platelet and white blood cell (WBC) separation efficiencies were quantified using a central composite factorial experimental design. These data were then fit using second order models. Both the model for the WBC separation efficiency and the model for the platelet separation efficiency predict that RPM has the greatest effect on separation efficiency and that RBC and plasma recycle have detrimental effects at moderate to low RPM, but have negligible impact on separation efficiency at high RPM.

Effects of Sample Impurities on the Analysis of MS2 Bacteriophage by Small-Angle Neutron Scattering

DTIC Science & Technology

2005-08-01

and the efficiency of water treatment plants and filtration devices (Jolis et al., 1999;3 Oppenheimer et al., 1997;4 Woolwine and Gerberding, 1995;5...the solvent water to deuterated water ratio so that structural information about the protein and nucleic acid components can be obtained separately...de-stained in a 30% methanol: 10% acetic acid:60% (v/v) water solution for 8 hr (Maniatis, Fritsch et al., 1982).37 2.4 SANS Measurements. SANS

Enhanced photocurrent production by bio-dyes of photosynthetic macromolecules on designed TiO2 film

PubMed Central

Yu, Daoyong; Wang, Mengfei; Zhu, Guoliang; Ge, Baosheng; Liu, Shuang; Huang, Fang

2015-01-01

The macromolecular pigment-protein complex has the merit of high efficiency for light-energy capture and transfer after long-term photosynthetic evolution. Here bio-dyes of A. platensis photosystem I (PSI) and spinach light-harvesting complex II (LHCII) are spontaneously sensitized on three types of designed TiO2 films, to assess the effects of pigment-protein complex on the performance of bio-dye sensitized solar cells (SSC). Adsorption models of bio-dyes are proposed based on the 3D structures of PSI and LHCII, and the size of particles and inner pores in the TiO2 film. PSI shows its merit of high efficiency for captured energy transfer, charge separation and transfer in the electron transfer chain (ETC), and electron injection from FB to the TiO2 conducting band. After optimization, the best short current (JSC) and photoelectric conversion efficiency (η) of PSI-SSC and LHCII-SSC are 1.31 mA cm-2 and 0.47%, and 1.51 mA cm-2 and 0.52%, respectively. The potential for further improvement of this PSI based SSC is significant and could lead to better utilization of solar energy. PMID:25790735

Signal Partitioning Algorithm for Highly Efficient Gaussian Mixture Modeling in Mass Spectrometry

PubMed Central

Polanski, Andrzej; Marczyk, Michal; Pietrowska, Monika; Widlak, Piotr; Polanska, Joanna

2015-01-01

Mixture - modeling of mass spectra is an approach with many potential applications including peak detection and quantification, smoothing, de-noising, feature extraction and spectral signal compression. However, existing algorithms do not allow for automated analyses of whole spectra. Therefore, despite highlighting potential advantages of mixture modeling of mass spectra of peptide/protein mixtures and some preliminary results presented in several papers, the mixture modeling approach was so far not developed to the stage enabling systematic comparisons with existing software packages for proteomic mass spectra analyses. In this paper we present an efficient algorithm for Gaussian mixture modeling of proteomic mass spectra of different types (e.g., MALDI-ToF profiling, MALDI-IMS). The main idea is automated partitioning of protein mass spectral signal into fragments. The obtained fragments are separately decomposed into Gaussian mixture models. The parameters of the mixture models of fragments are then aggregated to form the mixture model of the whole spectrum. We compare the elaborated algorithm to existing algorithms for peak detection and we demonstrate improvements of peak detection efficiency obtained by using Gaussian mixture modeling. We also show applications of the elaborated algorithm to real proteomic datasets of low and high resolution. PMID:26230717

ANALYSIS OF GLYCANS DERIVED FROM GLYCOCONJUGATES BY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

PubMed Central

Mechref, Yehia

2012-01-01

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful mass spectrometric (MS) and tandem MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high order tandem MS (MSn). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and gas chromatography (GC). However, capillary electrophoresis (CE) and microfluidics capillary electrophoresis (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to mass spectrometry. PMID:22180203

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

PubMed

Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M; Frick, David N; Schiffer, Celia A

2013-12-01

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo. © 2013 The Protein Society.

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Yan, Weiying; Sloat, Amy L; Yagi, Shigeyuki; Nakazumi, Hiroyuki; Colyer, Christa L

2006-04-01

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization.

PubMed

Alarcón, Emilio; Edwards, Ana Maria; Aspee, Alexis; Moran, Faustino E; Borsarelli, Claudio D; Lissi, Eduardo A; Gonzalez-Nilo, Danilo; Poblete, Horacio; Scaiano, J C

2010-01-01

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

PubMed

Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

2007-03-01

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

Pi-Pi contacts are an overlooked protein feature relevant to phase separation

PubMed Central

Vernon, Robert McCoy; Chong, Paul Andrew; Tsang, Brian; Kim, Tae Hun; Bah, Alaji; Farber, Patrick; Lin, Hong

2018-01-01

Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing. PMID:29424691

Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

PubMed

Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

2015-06-16

In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.

Blending protein separation and peptide analysis through real-time proteolytic digestion.

PubMed

Slysz, Gordon W; Schriemer, David C

2005-03-15

Typical liquid- or gel-based protein separations require enzymatic digestion as an important first step in generating protein identifications. Traditional protocols involve long-term proteolytic digestion of the separated protein, often leading to sample loss and reduced sensitivity. Previously, we presented a rapid method of proteolytic digestion that showed excellent digestion of resistant and low concentrations of protein without requiring reduction and alkylation. Here, we demonstrate on-line, real-time tryptic digestion in conjunction with reversed-phase protein separation. The studies were aimed at optimizing pH and ionic strength and the size of the digestion element, to produce maximal protein digestion with minimal effects on chromatographic integrity. Upon establishing optimal conditions, the digestion element was attached downstream from a capillary C4 reversed-phase column. A four-protein mixture was processed through the combined system, and the resulting peptides were analyzed on-line by electrospray mass spectrometry. Extracted ion chromatograms for protein chromatography based on peptide elution were generated. These were shown to emulate ion chromatograms produced in a subsequent run without the digestion element, based on protein elution. The methodology will enable rapid and sensitive analysis of liquid-based protein separations using the power of bottom-up proteomics methodologies.

Protein Separation by Electrophoretic-Electroosmotic Focusing on Supported Lipid Bilayers

PubMed Central

Liu, Chunming; Monson, Christopher F.; Yang, Tinglu; Pace, Hudson; Cremer, Paul S.

2011-01-01

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch. PMID:21958061

High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.

PubMed

Reddy, S G; Cochran, B J; Worth, L L; Knutson, V P; Haddox, M K

1994-04-01

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

Separation and recovery of fine particles from waste circuit boards using an inflatable tapered diameter separation bed.

PubMed

Duan, Chenlong; Sheng, Cheng; Wu, Lingling; Zhao, Yuemin; He, Jinfeng; Zhou, Enhui

2014-01-01

Recovering particle materials from discarded printed circuit boards can enhance resource recycling and reduce environmental pollution. Efficiently physically separating and recovering fine metal particles (-0.5 mm) from the circuit boards are a key recycling challenge. To do this, a new type of separator, an inflatable tapered diameter separation bed, was developed to study particle motion and separation mechanisms in the bed's fluid flow field. For 0.5-0.25 mm circuit board particles, metal recovery rates ranged from 87.56 to 94.17%, and separation efficiencies ranged from 87.71 to 94.20%. For 0.25-0.125 mm particles, metal recovery rates ranged from 84.76 to 91.97%, and separation efficiencies ranged from 84.74 to 91.86%. For superfine products (-0.125 mm), metal recovery rates ranged from 73.11 to 83.04%, and separation efficiencies ranged from 73.00 to 83.14%. This research showed that the inflatable tapered diameter separation bed achieved efficient particle separation and can be used to recover fine particles under a wide range of operational conditions. The bed offers a new mechanical technology to recycle valuable materials from discarded printed circuit boards, reducing environmental pollution.

Stochastic simulation of enzyme-catalyzed reactions with disparate timescales.

PubMed

Barik, Debashis; Paul, Mark R; Baumann, William T; Cao, Yang; Tyson, John J

2008-10-01

Many physiological characteristics of living cells are regulated by protein interaction networks. Because the total numbers of these protein species can be small, molecular noise can have significant effects on the dynamical properties of a regulatory network. Computing these stochastic effects is made difficult by the large timescale separations typical of protein interactions (e.g., complex formation may occur in fractions of a second, whereas catalytic conversions may take minutes). Exact stochastic simulation may be very inefficient under these circumstances, and methods for speeding up the simulation without sacrificing accuracy have been widely studied. We show that the "total quasi-steady-state approximation" for enzyme-catalyzed reactions provides a useful framework for efficient and accurate stochastic simulations. The method is applied to three examples: a simple enzyme-catalyzed reaction where enzyme and substrate have comparable abundances, a Goldbeter-Koshland switch, where a kinase and phosphatase regulate the phosphorylation state of a common substrate, and coupled Goldbeter-Koshland switches that exhibit bistability. Simulations based on the total quasi-steady-state approximation accurately capture the steady-state probability distributions of all components of these reaction networks. In many respects, the approximation also faithfully reproduces time-dependent aspects of the fluctuations. The method is accurate even under conditions of poor timescale separation.

Pi-Pi contacts are an overlooked protein feature relevant to phase separation.

PubMed

Vernon, Robert McCoy; Chong, Paul Andrew; Tsang, Brian; Kim, Tae Hun; Bah, Alaji; Farber, Patrick; Lin, Hong; Forman-Kay, Julie Deborah

2018-02-09

Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing. © 2018, Vernon et al.

Recyclability of PET/WPI/PE Multilayer Films by Removal of Whey Protein Isolate-Based Coatings with Enzymatic Detergents

PubMed Central

Cinelli, Patrizia; Schmid, Markus; Bugnicourt, Elodie; Coltelli, Maria Beatrice; Lazzeri, Andrea

2016-01-01

Multilayer plastic films provide a range of properties, which cannot be obtained from monolayer films but, at present, their recyclability is an open issue and should be improved. Research to date has shown the possibility of using whey protein as a layer material with the property of acting as an excellent barrier against oxygen and moisture, replacing petrochemical non-recyclable materials. The innovative approach of the present research was to achieve the recyclability of the substrate films by separating them, with a simple process compatible with industrial procedures, in order to promote recycling processes leading to obtain high value products that will beneficially impact the packaging and food industries. Hence, polyethyleneterephthalate (PET)/polyethylene (PE) multi-layer film was prepared based on PET coated with a whey protein layer, and then the previous structure was laminated with PE. Whey proteins, constituting the coating, can be degraded by enzymes so that the coating films can be washed off from the plastic substrate layer. Enzyme types, dosage, time, and temperature optima, which are compatible with procedures adopted in industrial waste recycling, were determined for a highly-efficient process. The washing of samples based on PET/whey and PET/whey/PE were efficient when performed with enzymatic detergent containing protease enzymes, as an alternative to conventional detergents used in recycling facilities. Different types of enzymatic detergents tested presented positive results in removing the protein layer from the PET substrate and from the PET/whey/PE multilayer films at room temperature. These results attested to the possibility of organizing the pre-treatment of the whey-based multilayer film by washing with different available commercial enzymatic detergents in order to separate PET and PE, thus allowing a better recycling of the two different polymers. Mechanical properties of the plastic substrate, such as stress at yield, stress and elongation at break, evaluated by tensile testing on films before and after cleaning, were are not significantly affected by washing with enzymatic detergents. PMID:28773592

Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

PubMed Central

Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

2015-01-01

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

Electrostatic separation for recycling conductors, semiconductors, and nonconductors from electronic waste.

PubMed

Xue, Mianqiang; Yan, Guoqing; Li, Jia; Xu, Zhenming

2012-10-02

Electrostatic separation has been widely used to separate conductors and nonconductors for recycling e-waste. However, the components of e-waste are complex, which can be classified as conductors, semiconductors, and nonconductors according to their conducting properties. In this work, we made a novel attempt to recover the mixtures containing conductors (copper), semiconductors (extrinsic silicon), and nonconductors (woven glass reinforced resin) by electrostatic separation. The results of binary mixtures separation show that the separation of conductor and nonconductor, semiconductor and nonconductor need a higher voltage level while the separation of conductor and semiconductor needs a higher roll speed. Furthermore, the semiconductor separation efficiency is more sensitive to the high voltage level and the roll speed than the conductor separation efficiency. An integrated process was proposed for the multiple mixtures separation. The separation efficiency of conductors and semiconductors can reach 82.5% and 88%, respectively. This study contributes to the efficient recycling of valuable resources from e-waste.

Compost biodegradation of recalcitrant hoof keratin by bacteria and fungi.

PubMed

Reuter, T; Gilroyed, B H; Xu, W; McAllister, T A; Stanford, K

2015-08-01

Compost activities efficiently break down a wide range of organic substances over time. In this study, bovine hoof was used as recalcitrant protein model to gain so far cryptic information on biodegradation during livestock mortalities composting. Bovine hooves (black and white), containing different amounts of melanin, placed into nylon bags were monitored during composting of cattle mortalities for up to 230 days. Besides physiochemical analysis, bacterial 16S and fungal 18S DNA fragments were amplified by PCR and profiles were separated by DGGE. Sequence analysis of separated fragments revealed various bacterial and fungal identities during composting. The microbial diversity was affected by a time-temperature interaction and by the hoof colour. Our molecular data, supported by electron microscopy, suggest hoof colonization by shifting bacteria and fungi communities. During composting, microbial communities work collaboratively in the degradation of recalcitrant organic matter such as keratin over time. A number of biomolecules including recalcitrant proteins may persist in environmental reservoirs, but breakdown can occur during composting. A combination of bioactivity and physiochemical conditions appear to be decisive for the fate of persistent biomolecules. © 2015 The Society for Applied Microbiology.

[Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry].

PubMed

Li, Feng; Kang, Jingwu

2014-04-01

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.

Quantum coherence spectroscopy reveals complex dynamics in bacterial light-harvesting complex 2 (LH2).

PubMed

Harel, Elad; Engel, Gregory S

2012-01-17

Light-harvesting antenna complexes transfer energy from sunlight to photosynthetic reaction centers where charge separation drives cellular metabolism. The process through which pigments transfer excitation energy involves a complex choreography of coherent and incoherent processes mediated by the surrounding protein and solvent environment. The recent discovery of coherent dynamics in photosynthetic light-harvesting antennae has motivated many theoretical models exploring effects of interference in energy transfer phenomena. In this work, we provide experimental evidence of long-lived quantum coherence between the spectrally separated B800 and B850 rings of the light-harvesting complex 2 (LH2) of purple bacteria. Spectrally resolved maps of the detuning, dephasing, and the amplitude of electronic coupling between excitons reveal that different relaxation pathways act in concert for optimal transfer efficiency. Furthermore, maps of the phase of the signal suggest that quantum mechanical interference between different energy transfer pathways may be important even at ambient temperature. Such interference at a product state has already been shown to enhance the quantum efficiency of transfer in theoretical models of closed loop systems such as LH2.

Quantum coherence spectroscopy reveals complex dynamics in bacterial light-harvesting complex 2 (LH2)

PubMed Central

Harel, Elad; Engel, Gregory S.

2012-01-01

Light-harvesting antenna complexes transfer energy from sunlight to photosynthetic reaction centers where charge separation drives cellular metabolism. The process through which pigments transfer excitation energy involves a complex choreography of coherent and incoherent processes mediated by the surrounding protein and solvent environment. The recent discovery of coherent dynamics in photosynthetic light-harvesting antennae has motivated many theoretical models exploring effects of interference in energy transfer phenomena. In this work, we provide experimental evidence of long-lived quantum coherence between the spectrally separated B800 and B850 rings of the light-harvesting complex 2 (LH2) of purple bacteria. Spectrally resolved maps of the detuning, dephasing, and the amplitude of electronic coupling between excitons reveal that different relaxation pathways act in concert for optimal transfer efficiency. Furthermore, maps of the phase of the signal suggest that quantum mechanical interference between different energy transfer pathways may be important even at ambient temperature. Such interference at a product state has already been shown to enhance the quantum efficiency of transfer in theoretical models of closed loop systems such as LH2. PMID:22215585

Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

PubMed

Noah, Alexandre Mboene; Niemenak, Nicolas; Sunderhaus, Stephanie; Haase, Christin; Omokolo, Denis Ndoumou; Winkelmann, Traud; Braun, Hans-Peter

2013-01-14

Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Native red electrophoresis--a new method suitable for separation of native proteins.

PubMed

Dráb, Tomáš; Kračmerová, Jana; Tichá, Ivana; Hanzlíková, Eva; Tichá, Marie; Ryšlavá, Helena; Doubnerová, Veronika; Maňásková-Postlerová, Pavla; Liberda, Jiří

2011-12-01

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of a commercial electro-kinetically pumped sheath-flow nanospray interface coupled to an automated capillary zone electrophoresis system.

PubMed

Peuchen, Elizabeth H; Zhu, Guije; Sun, Liangliang; Dovichi, Norman J

2017-03-01

Capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) is attracting renewed attention for proteomic and metabolomic analysis. An important reason for this interest is the maturation and commercialization of interfaces for coupling CZE with ESI-MS. One of these interfaces is an electro-kinetically pumped sheath flow nanospray interface developed by the Dovichi group, in which a very low sheath flow is generated based on electroosmosis within a glass emitter. CMP Scientific has commercialized this interface as the EMASS-II ion source. In this work, we compared the performance of the EMASS-II ion source with our in-house system. The performance of the systems is equivalent. We also coupled the EMASS-II ion source with a PrinCE Next|480 capillary electrophoresis autosampler and an Orbitrap mass spectrometer, and analyzed this system's performance in terms of sensitivity, reproducibility, and separation performance for separation of tryptic digests, intact proteins, and amino acids. The system produced reproducible analysis of BSA digest; the RSDs of peptide intensity and migration time across 24 runs were less than 20 and 6%, respectively. The system produced a linear calibration curve of intensity across a 30-fold range of tryptic digest concentration. The combination of a commercial autosampler and electrospray interface efficiently separated amino acids, peptides, and intact proteins, and only required 5 μL of sample for analysis. Graphical Abstract The commercial and locally constructed versions of the interface provide similar numbers of protein identifications from a Xenopus laevis fertilized egg digest.

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

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

PubMed

Hanrieder, Jörg; Zuberovic, Aida; Bergquist, Jonas

2009-04-24

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

Effects of ammonium sulfate and sodium chloride concentration on PEG/protein liquid-liquid phase separation.

PubMed

Dumetz, André C; Lewus, Rachael A; Lenhoff, Abraham M; Kaler, Eric W

2008-09-16

When added to protein solutions, poly(ethylene glycol) (PEG) creates an effective attraction between protein molecules due to depletion forces. This effect has been widely used to crystallize proteins, and PEG is among the most successful crystallization agents in current use. However, PEG is almost always used in combination with a salt at either low or relatively high concentrations. Here the effects of sodium chloride and ammonium sulfate concentration on PEG 8000/ovalbumin liquid-liquid (L-L) phase separation are investigated. At low salt the L-L phase separation occurs at decreasing protein concentration with increasing salt concentration, presumably due to repulsive electrostatic interactions between proteins. At high salt concentration, the behavior depends on the nature of the salt. Sodium chloride has little effect on the L-L phase separation, but ammonium sulfate decreases the protein concentration at which the L-L phase separation occurs. This trend is attributed to the effects of critical fluctuations on depletion forces. The implications of these results for designing solution conditions optimal for protein crystallization are discussed.

Factors affecting the separation performance of proteins in capillary electrophoresis.

PubMed

Zhu, Yueping; Li, Zhenqing; Wang, Ping; Shen, Lisong; Zhang, Dawei; Yamaguchi, Yoshinori

2018-04-15

Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min. Copyright © 2018 Elsevier B.V. All rights reserved.

Separation of transition metals on a poly-iminodiacetic acid grafted polymeric resin column with post-column reaction detection utilising a paired emitter-detector diode system.

PubMed

Barron, Leon; O'Toole, Martina; Diamond, Dermot; Nesterenko, Pavel N; Paull, Brett

2008-12-05

The selectivity, retention and separation of transition metals on a short (2 mm x 50 mm) column packed with a poly-iminodiacetic acid functionalised polymer 10 microm resin (Dionex ProPac IMAC-10) are presented. This stationary phase, typically used for the separation of proteins, is composed of long chain poly-iminodiacetic acid groups grafted to a hydrophilic layer surrounding a 10 microm polymeric bead. Through the use of a combination of a multi-step pH and picolinic acid gradient, the separation of magnesium, iron, cobalt, cadmium, zinc, lead and copper was possible, followed by post-column reaction with 4-(2-pyridylazo) resorcinol (PAR) and absorbance detection at 510 nm using a novel and inexpensive optical detector, comprised of two light emitting diodes with one acting as a light source and the other as a detector. Column efficiency for selective transition metals was in excess of N=10,000, with the baseline separation of seven metal cations in <3 min possible under optimised conditions. Detection limits of between 5 and 81 microg/L were possible based upon a 50 microL injection volume.

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

NASA Astrophysics Data System (ADS)

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Salaklang, Jatuporn; Hofmann, Heinrich

2014-05-01

Because of their biocompatibility and unique magnetic properties, superparamagnetic iron oxide nanoparticles NPs (SPIONs) are recognized as some of the most prominent agents for theranostic applications. Thus, understanding the interaction of SPIONs with biological systems is important for their safe design and efficient applications. In this study, SPIONs were coated with 2 different polymers: polyvinyl alcohol polymer (PVA) and dextran. The obtained NPs with different surface charges (positive, neutral, and negative) were used as a model study of the effect of surface charges and surface polymer materials on protein adsorption using a magnetic separator. We found that the PVA-coated SPIONs with negative and neutral surface charge adsorbed more serum proteins than the dextran-coated SPIONs, which resulted in higher blood circulation time for PVA-coated NPs than the dextran-coated ones. Highly abundant proteins such as serum albumin, serotransferrin, prothrombin, alpha-fetoprotein, and kininogen-1 were commonly found on both PVA- and dextran-coated SPIONs. By increasing the ionic strength, soft- and hard-corona proteins were observed on 3 types of PVA-SPIONs. However, the tightly bound proteins were observed only on negatively charged PVA-coated SPIONs after the strong protein elution.

Recent advances on multidimensional liquid chromatography-mass spectrometry for proteomics: from qualitative to quantitative analysis--a review.

PubMed

Wu, Qi; Yuan, Huiming; Zhang, Lihua; Zhang, Yukui

2012-06-20

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography-mass spectrometry (MDLC-MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including "top-down" and "bottom-up" to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes. Copyright © 2012 Elsevier B.V. All rights reserved.

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

PubMed

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

2012-12-01

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

A computational model of pattern separation efficiency in the dentate gyrus with implications in schizophrenia

PubMed Central

Faghihi, Faramarz; Moustafa, Ahmed A.

2015-01-01

Information processing in the hippocampus begins by transferring spiking activity of the entorhinal cortex (EC) into the dentate gyrus (DG). Activity pattern in the EC is separated by the DG such that it plays an important role in hippocampal functions including memory. The structural and physiological parameters of these neural networks enable the hippocampus to be efficient in encoding a large number of inputs that animals receive and process in their life time. The neural encoding capacity of the DG depends on its single neurons encoding and pattern separation efficiency. In this study, encoding by the DG is modeled such that single neurons and pattern separation efficiency are measured using simulations of different parameter values. For this purpose, a probabilistic model of single neurons efficiency is presented to study the role of structural and physiological parameters. Known neurons number of the EC and the DG is used to construct a neural network by electrophysiological features of granule cells of the DG. Separated inputs as activated neurons in the EC with different firing probabilities are presented into the DG. For different connectivity rates between the EC and DG, pattern separation efficiency of the DG is measured. The results show that in the absence of feedback inhibition on the DG neurons, the DG demonstrates low separation efficiency and high firing frequency. Feedback inhibition can increase separation efficiency while resulting in very low single neuron’s encoding efficiency in the DG and very low firing frequency of neurons in the DG (sparse spiking). This work presents a mechanistic explanation for experimental observations in the hippocampus, in combination with theoretical measures. Moreover, the model predicts a critical role for impaired inhibitory neurons in schizophrenia where deficiency in pattern separation of the DG has been observed. PMID:25859189

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

PubMed

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis.

PubMed

Ahn, Sang-Joon; Burne, Robert A

2006-10-01

The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

Key factors of eddy current separation for recovering aluminum from crushed e-waste.

PubMed

Ruan, Jujun; Dong, Lipeng; Zheng, Jie; Zhang, Tao; Huang, Mingzhi; Xu, Zhenming

2017-02-01

Recovery of e-waste in China had caused serious pollutions. Eddy current separation is an environment-friendly technology of separating nonferrous metallic particles from crushed e-waste. However, due to complex particle characters, separation efficiency of traditional eddy current separator was low. In production, controllable operation factors of eddy current separation are feeding speed, (ωR-v), and S p . There is little special information about influencing mechanism and critical parameters of these factors in eddy current separation. This paper provided the special information of these key factors in eddy current separation of recovering aluminum particles from crushed waste refrigerator cabinets. Detachment angles increased as the increase of (ωR-v). Separation efficiency increased with the growing of detachment angles. Aluminum particles were completely separated from plastic particles in critical parameters of feeding speed 0.5m/s and detachment angles greater than 6.61deg. S p /S m of aluminum particles in crushed waste refrigerators ranged from 0.08 to 0.51. Separation efficiency increased as the increase of S p /S m . This enlightened us to develop new separator to separate smaller nonferrous metallic particles in e-waste recovery. High feeding speed destroyed separation efficiency. However, greater S p of aluminum particles brought positive impact on separation efficiency. Greater S p could increase critical feeding speed to offer greater throughput of eddy current separation. This paper will guide eddy current separation in production of recovering nonferrous metals from crushed e-waste. Copyright © 2016 Elsevier Ltd. All rights reserved.

Separation and Recovery of Fine Particles from Waste Circuit Boards Using an Inflatable Tapered Diameter Separation Bed

PubMed Central

Sheng, Cheng; Wu, Lingling; Zhao, Yuemin; He, Jinfeng; Zhou, Enhui

2014-01-01

Recovering particle materials from discarded printed circuit boards can enhance resource recycling and reduce environmental pollution. Efficiently physically separating and recovering fine metal particles (−0.5 mm) from the circuit boards are a key recycling challenge. To do this, a new type of separator, an inflatable tapered diameter separation bed, was developed to study particle motion and separation mechanisms in the bed's fluid flow field. For 0.5–0.25 mm circuit board particles, metal recovery rates ranged from 87.56 to 94.17%, and separation efficiencies ranged from 87.71 to 94.20%. For 0.25–0.125 mm particles, metal recovery rates ranged from 84.76 to 91.97%, and separation efficiencies ranged from 84.74 to 91.86%. For superfine products (−0.125 mm), metal recovery rates ranged from 73.11 to 83.04%, and separation efficiencies ranged from 73.00 to 83.14%. This research showed that the inflatable tapered diameter separation bed achieved efficient particle separation and can be used to recover fine particles under a wide range of operational conditions. The bed offers a new mechanical technology to recycle valuable materials from discarded printed circuit boards, reducing environmental pollution. PMID:25379546

The relative vertex clustering value - a new criterion for the fast discovery of functional modules in protein interaction networks

PubMed Central

2015-01-01

Background Cellular processes are known to be modular and are realized by groups of proteins implicated in common biological functions. Such groups of proteins are called functional modules, and many community detection methods have been devised for their discovery from protein interaction networks (PINs) data. In current agglomerative clustering approaches, vertices with just a very few neighbors are often classified as separate clusters, which does not make sense biologically. Also, a major limitation of agglomerative techniques is that their computational efficiency do not scale well to large PINs. Finally, PIN data obtained from large scale experiments generally contain many false positives, and this makes it hard for agglomerative clustering methods to find the correct clusters, since they are known to be sensitive to noisy data. Results We propose a local similarity premetric, the relative vertex clustering value, as a new criterion allowing to decide when a node can be added to a given node's cluster and which addresses the above three issues. Based on this criterion, we introduce a novel and very fast agglomerative clustering technique, FAC-PIN, for discovering functional modules and protein complexes from a PIN data. Conclusions Our proposed FAC-PIN algorithm is applied to nine PIN data from eight different species including the yeast PIN, and the identified functional modules are validated using Gene Ontology (GO) annotations from DAVID Bioinformatics Resources. Identified protein complexes are also validated using experimentally verified complexes. Computational results show that FAC-PIN can discover functional modules or protein complexes from PINs more accurately and more efficiently than HC-PIN and CNM, the current state-of-the-art approaches for clustering PINs in an agglomerative manner. PMID:25734691

Aptamer functionalized noble metal particles for bioanalytical and biomedical applications

NASA Astrophysics Data System (ADS)

Yasun, Emir

Noble metal particles, especially gold (Au) and silver (Ag) have been exploited in a broad range of biological applications due to their unique intrinsic features that depend on their physical appearance or optoelectronic properties, which can be tuned with the change in the size or shape of those particles. Thus, this tunability enables gold nanoparticles (AuNPs) to be used in biomedical diagnostic and therapeutical applications. In photothermal therapy applications, nanomaterials, which can absorb efficiently in NIR region, are utilized since the healthy tissue or cells can't absorb at this spectral region. Among AuNPs, gold nanorods (AuNRs) are one of the best candidates for hyperthermia therapy of cancer cells with their high absorption cross-sections and tunable absorption maxima in NIR region. When this unique optical property is combined with the specificity against cancer cells utilized by aptamer conjugations, AuNRs become to be one of the most important nanoparticles employed in both cancer cell sensing and therapy. However, one drawback of AuNRs is having the surfactant CTAB on their surface, which can cause nonspecificity and cytotoxicity. In this research, the side effects of CTAB are passivated by BSA modification, where the nonspecificity and cytotoxicity are dramatically decreased prior to the NIR treatment. Recognition of changes in the rare cancer protein abundances can lead the early diagnosis of cancer, so capturing these low abundance proteins has a great significance. In this research, firstly, aptamer conjugated AuNRs were used to capture 1ng of a-thrombin effectively from plasma samples as model system. Then both aptamer conjugated AuNRs and silver microspheres (SMSs) are used to capture the biomarker proteins of a colon cancer cell line, DLD-1. Gold and silver surfaces can easily be modified through thiolate chemistry, compared to the tedious modification steps for the magnetic particles, so more aptamer immobilization can be achieved for AuNRs and SMSs, which can increase the possibility of binding to the target protein. Furthermore, SMSs offer a novel separation method, gravitational separation owing to their heavy nature. In this way, there is no need for an external stimuli to separate the captured proteins and protein isolation can take only seconds.

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Recycling of rare earth particle by mini-hydrocyclones.

PubMed

Yu, Jian-Feng; Fu, Jian; Cheng, Hao; Cui, Zhengwei

2017-03-01

Mini-hydrocyclones were applied to separate the fine rare earth particles from the suspensions. The effects of the flow rate, split ratio, and feed concentration on the total separation efficiency and grade separation efficiency were studied. The combined effects of the flow rate (1200-1600L/h), split ratio (20-60%) and concentration (0.6-1.0wt%) on the total separation efficiency in mini-hydrocyclones were investigated using a response surface methodology. The optimum operating parameters for a total separation efficiency of 92.5% were: feed flow rate=1406L/h, split ratio=20%, and feed concentration=1wt%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pharmaceutical Perspective on Opalescence and Liquid-Liquid Phase Separation in Protein Solutions.

PubMed

Raut, Ashlesha S; Kalonia, Devendra S

2016-05-02

Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations. A brief discussion on theoretical concepts based on thermodynamics, kinetics, and light scattering is presented. This review also discusses theoretical concepts behind intense light scattering in the vicinity of the critical point termed as "critical opalescence". Both opalescence and LLPS are affected by the formulation factors including pH, ionic strength, protein concentration, temperature, and excipients. Literature reports for the effect of these formulation factors on attractive protein-protein interactions in solution as assessed by the second virial coefficient (B2) and the cloud-point temperature (Tcloud) measurements are also presented. The review also highlights pharmaceutical implications of LLPS in protein solutions.

Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response

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

Riback, Joshua A.; Katanski, Christopher D.; Kear-Scott, Jamie L.

In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1’s LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we createmore » LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.« less

Modulator Dynamics Shape the Design Space for Stepwise-Elution Simulated Moving Bed Chromatographic Separations.

PubMed

Wayne, Chris J; Velayudhan, Ajoy

2018-03-31

For proteins and other biological macromolecules, SMB chromatography is best operated non-isocratically. However, traditional modes of non-isocratic SMB operation generate significant mobile-phase modulator dynamics. The mechanisms by which these modulator dynamics affect a separation's success, and thus frame the design space, have yet to be explained quantitatively. Here, the dynamics of the modulator (e.g., salts in ion exchange and hydrophobic interaction chromatography) are explicitly accounted for. This leads to the elucidation of two new design constraints, presented as dimensionless numbers, which quantify the effects of the modulator phenomena and thus predict the success of a non-isocratic SMB separation. Consequently, these two new design constraints re-define the SMB design space. Computational and experimental studies at the boundaries of this design space corroborate the theoretical predictions. The design of efficient and robust operating conditions through use of the new design space is also demonstrated. © 2018 The Authors. Biotechnology Journal Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations.

PubMed

Schmidt, Thomas H; Kandt, Christian

2012-10-22

At the beginning of each molecular dynamics membrane simulation stands the generation of a suitable starting structure which includes the working steps of aligning membrane and protein and seamlessly accommodating the protein in the membrane. Here we introduce two efficient and complementary methods based on pre-equilibrated membrane patches, automating these steps. Using a voxel-based cast of the coarse-grained protein, LAMBADA computes a hydrophilicity profile-derived scoring function based on which the optimal rotation and translation operations are determined to align protein and membrane. Employing an entirely geometrical approach, LAMBADA is independent from any precalculated data and aligns even large membrane proteins within minutes on a regular workstation. LAMBADA is the first tool performing the entire alignment process automatically while providing the user with the explicit 3D coordinates of the aligned protein and membrane. The second tool is an extension of the InflateGRO method addressing the shortcomings of its predecessor in a fully automated workflow. Determining the exact number of overlapping lipids based on the area occupied by the protein and restricting expansion, compression and energy minimization steps to a subset of relevant lipids through automatically calculated and system-optimized operation parameters, InflateGRO2 yields optimal lipid packing and reduces lipid vacuum exposure to a minimum preserving as much of the equilibrated membrane structure as possible. Applicable to atomistic and coarse grain structures in MARTINI format, InflateGRO2 offers high accuracy, fast performance, and increased application flexibility permitting the easy preparation of systems exhibiting heterogeneous lipid composition as well as embedding proteins into multiple membranes. Both tools can be used separately, in combination with other methods, or in tandem permitting a fully automated workflow while retaining a maximum level of usage control and flexibility. To assess the performance of both methods, we carried out test runs using 22 membrane proteins of different size and transmembrane structure.

Protein separation using an electrically tunable membrane

NASA Astrophysics Data System (ADS)

Jou, Ining; Melnikov, Dmitriy; Gracheva, Maria

Separation of small proteins by charge with a solid-state porous membrane requires control over the protein's movement. Semiconductor membrane has this ability due to the electrically tunable electric potential profile inside the nanopore. In this work we investigate the possibility to separate the solution of two similar sized proteins by charge. As an example, we consider two small globular proteins abundant in humans: insulin (negatively charged) and ubiquitin (neutral). We find that the localized electric field inside the pore either attracts or repels the charged protein to or from the pore wall which affects the delay time before a successful translocation of the protein through the nanopore. However, the motion of the uncharged ubiquitin is unaffected. The difference in the delay time (and hence the separation) can be further increased by the application of the electrolyte bias which induces an electroosmotic flow in the pore. NSF DMR and CBET Grant No. 1352218.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue.

PubMed

Pennington, Kyla; McGregor, Emma; Beasley, Clare L; Everall, Ian; Cotter, David; Dunn, Michael J

2004-01-01

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Electrostatic separation for recycling silver, silicon and polyethylene terephthalate from waste photovoltaic cells

NASA Astrophysics Data System (ADS)

Zhang, Zisheng; Sun, Bo; Yang, Jie; Wei, Yusheng; He, Shoujie

2017-04-01

Electrostatic separation technology has been proven to be an effective and environmentally friendly way of recycling electronic waste. In this study, this technology was applied to recycle waste solar panels. Mixed particles of silver and polyethylene terephthalate, silicon and polyethylene terephthalate, and silver and silicon were separated with a single-roll-type electrostatic separator. The influence of high voltage level, roll speed, radial position corona electrode and angular position of the corona electrode on the separation efficiency was studied. The experimental data showed that separation of silver/polyethylene terephthalate and silicon/polyethylene terephthalate needed a higher voltage level, while separation of silver and silicon needed a smaller angular position for the corona electrode and a higher roll speed. The change of the high voltage level, roll speed, radial position of the corona electrode, and angular position of the corona electrode has more influence on silicon separation efficiency than silver separation efficiency. An integrated process is proposed using a two-roll-type corona separator for multistage separation of a mixture of these three materials. The separation efficiency for silver and silicon were found to reach 96% and 98%, respectively.

Using Biomolecules to Separate Plutonium

NASA Astrophysics Data System (ADS)

Gogolski, Jarrod

Used nuclear fuel has traditionally been treated through chemical separations of the radionuclides for recycle or disposal. This research considers a biological approach to such separations based on a series of complex and interdependent interactions that occur naturally in the human body with plutonium. These biological interactions are mediated by the proteins serum transferrin and the transferrin receptor. Transferrin to plutonium in vivo and can deposit plutonium into cells after interacting with the transferrin receptor protein at the cell surface. Using cerium as a non-radioactive surrogate for plutonium, it was found that cerium(IV) required multiple synergistic anions to bind in the N-lobe of the bilobal transferrin protein, creating a conformation of the cerium-loaded protein that would be unable to interact with the transferrin receptor protein to achieve a separation. The behavior of cerium binding to transferrin has contributed to understanding how plutonium(IV)-transferrin interacts in vivo and in biological separations.

Evaluation of an ODS column modified with zwitterionic/nonionic mixed surfactants and its application to direct injection determination of inorganic anions.

PubMed

Hasegawa, Takuya; Umemura, Tomonari; Koide, Akira; Chiba, Koichi; Ueki, Yuji; Tsunoda, Kin-ichi; Haraguchi, Hiroki

2005-08-01

An octadecylsilica (ODS) column modified with zwitterionic/nonionic mixed surfactants was evaluated for the direct injection determination of inorganic anions in biological fluids by ion chromatography. A zwitterionic surfactant (sulfobetaine-type) and a nonionic surfactant (polyoxyethylene-type) were used for a stationary-phase modification. When aqueous electrolyte solutions with concentrations of sub-mM to several mM were used as a mobile phase, the zwitterionic surfactant coated on the ODS surface exhibited unique separation selectivity for ionic species, while the nonionic surfactant coated on the ODS might have formed a hydrophilic network over the ODS surface and restricted matrix proteins from adsorbing on the stationary phase. Consequently, the mixed surfactant-modified column system allowed an efficient ion chromatographic separation of inorganic anions as well as a size-exclusive removal of column-fouling proteins. This separation system was applied to the direct injection determination of UV-absorbing anions in human saliva. The detection limits for nitrite, nitrate, iodide and thiocyanate were 3.1, 2.7, 4.5 and 25 microM, respectively, with UV detection at 210 nm (injection volume; 20 microl), and their relative standard deviations for 5 replicate measurements of saliva samples spiked with 100 microM each of those anions were 1.4, 0.9, 2.2 and 5.5%, respectively.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

PubMed

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Liquid-Liquid Phase Separation in a Dual Variable Domain Immunoglobulin Protein Solution: Effect of Formulation Factors and Protein-Protein Interactions.

PubMed

Raut, Ashlesha S; Kalonia, Devendra S

2015-09-08

Dual variable domain immunoglobulin proteins (DVD-Ig proteins) are large molecules (MW ∼ 200 kDa) with increased asymmetry because of their extended Y-like shape, which results in increased formulation challenges. Liquid-liquid phase separation (LLPS) of protein solutions into protein-rich and protein-poor phases reduces solution stability at intermediate concentrations and lower temperatures, and is a serious concern in formulation development as therapeutic proteins are generally stored at refrigerated conditions. In the current work, LLPS was studied for a DVD-Ig protein molecule as a function of solution conditions by measuring solution opalescence. LLPS of the protein was confirmed by equilibrium studies and by visually observing under microscope. The protein does not undergo any structural change after phase separation. Protein-protein interactions were measured by light scattering (kD) and Tcloud (temperature that marks the onset of phase separation). There is a good agreement between kD measured in dilute solution with Tcloud measured in the critical concentration range. Results indicate that the increased complexity of the molecule (with respect to size, shape, and charge distribution on the molecule) increases contribution of specific and nonspecific interactions in solution, which are affected by formulation factors, resulting in LLPS for DVD-Ig protein.

[Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

PubMed

Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

2017-10-01

In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

Accounting for host cell protein behavior in anion-exchange chromatography.

PubMed

Swanson, Ryan K; Xu, Ruo; Nettleton, Daniel S; Glatz, Charles E

2016-11-01

Host cell proteins (HCP) are a problematic set of impurities in downstream processing (DSP) as they behave most similarly to the target protein during separation. Approaching DSP with the knowledge of HCP separation behavior would be beneficial for the production of high purity recombinant biologics. Therefore, this work was aimed at characterizing the separation behavior of complex mixtures of HCP during a commonly used method: anion-exchange chromatography (AEX). An additional goal was to evaluate the performance of a statistical methodology, based on the characterization data, as a tool for predicting protein separation behavior. Aqueous two-phase partitioning followed by two-dimensional electrophoresis provided data on the three physicochemical properties most commonly exploited during DSP for each HCP: pI (isoelectric point), molecular weight, and surface hydrophobicity. The protein separation behaviors of two alternative expression host extracts (corn germ and E. coli) were characterized. A multivariate random forest (MVRF) statistical methodology was then applied to the database of characterized proteins creating a tool for predicting the AEX behavior of a mixture of proteins. The accuracy of the MVRF method was determined by calculating a root mean squared error value for each database. This measure never exceeded a value of 0.045 (fraction of protein populating each of the multiple separation fractions) for AEX. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1453-1463, 2016. © 2016 American Institute of Chemical Engineers.

Separation efficiency of the MASHA facility for short-lived mercury isotopes

NASA Astrophysics Data System (ADS)

Rodin, A. M.; Belozerov, A. V.; Chernysheva, E. V.; Dmitriev, S. N.; Gulyaev, A. V.; Gulyaeva, A. V.; Itkis, M. G.; Kliman, J.; Kondratiev, N. A.; Krupa, L.; Novoselov, A. S.; Oganessian, Yu. Ts.; Podshibyakin, A. V.; Salamatin, V. S.; Siváček, I.; Stepantsov, S. V.; Vanin, D. V.; Vedeneev, V. Yu.; Yukhimchuk, S. A.; Granja, C.; Pospisil, S.

2014-06-01

The mass-separator MASHA built to identify Super Heavy Elements by their mass-to-charge ratios is described. The results of the off- and on-line measurements of its separation efficiency are presented. In the former case four calibrated leaks of noble gases were used. In the latter the efficiency was measured via 284 MeV Ar beam and with using the hot catcher. The ECR ion source was used in both cases. The -radioactive isotopes of mercury produced in the complete fusion reaction Ar+SmHg+xn were detected at the mass-separator focal plane. The half-lives and the separation efficiency for the short-lived mercury isotopes were measured. Potentialities of the MEDIPIX detector system have been demonstrated for future use at the mass-separator MASHA.

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

Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

PubMed Central

Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

2012-01-01

Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma.

PubMed

Clifton, James; Huang, Feilei; Gaso-Sokac, Dajana; Brilliant, Kate; Hixson, Douglas; Josic, Djuro

2010-01-03

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

Miniaturized protein separation using a liquid chromatography column on a flexible substrate

NASA Astrophysics Data System (ADS)

Yang, Yongmo; Chae, Junseok

2008-12-01

We report a prototype protein separator that successfully miniaturizes existing technology for potential use in biocompatible health monitoring implants. The prototype is a liquid chromatography (LC) column (LC mini-column) fabricated on an inexpensive, flexible, biocompatible polydimethylsiloxane (PDMS) enclosure. The LC mini-column separates a mixture of proteins using size exclusion chromatography (SEC) with polydivinylbenzene beads (5-20 µm in diameter with 10 nm pore size). The LC mini-column is smaller than any commercially available LC column by a factor of ~11 000 and successfully separates denatured and native protein mixtures at ~71 psi of the applied fluidic pressure. Separated proteins are analyzed using NuPAGE-gel electrophoresis, high-performance liquid chromatography (HPLC) and an automated electrophoresis system. Quantitative HPLC results demonstrate successful separation based on intensity change: within 12 min, the intensity between large and small protein peaks changed by a factor of ~20. In further evaluation using the automated electrophoresis system, the plate height of the LC mini-column is between 36 µm and 100 µm. The prototype LC mini-column shows the potential for real-time health monitoring in applications that require inexpensive, flexible implant technology that can function effectively under non-laboratory conditions.

Rapid isolation of blood plasma using a cascaded inertial microfluidic device

PubMed Central

Robinson, M.; Hinsdale, T.; Coté, G.

2017-01-01

Blood, saliva, mucus, sweat, sputum, and other biological fluids are often hindered in their ability to be used in point-of-care (POC) diagnostics because their assays require some form of off-site sample pre-preparation to effectively separate biomarkers from larger components such as cells. The rapid isolation, identification, and quantification of proteins and other small molecules circulating in the blood plasma from larger interfering molecules are therefore particularly important factors for optical blood diagnostic tests, in particular, when using optical approaches that incur spectroscopic interference from hemoglobin-rich red blood cells (RBCs). In this work, a sequential spiral polydimethylsiloxane (PDMS) microfluidic device for rapid (∼1 min) on-chip blood cell separation is presented. The chip utilizes Dean-force induced migration via two 5-loop Archimedean spirals in series. The chip was characterized in its ability to filter solutions containing fluorescent beads and silver nanoparticles and further using blood solutions doped with a fluorescent protein. Through these experiments, both cellular and small molecule behaviors in the chip were assessed. The results exhibit an average RBC separation efficiency of ∼99% at a rate of 5.2 × 106 cells per second while retaining 95% of plasma components. This chip is uniquely suited for integration within a larger point-of-care diagnostic system for the testing of blood plasma, and the use of multiple filtering spirals allows for the tuning of filtering steps, making this device and the underlying technique applicable for a wide range of separation applications. PMID:28405258

Applicability of Separation Potentials to Determining the Parameters of Cascade Efficiency in Enrichment of Ternary Mixtures

NASA Astrophysics Data System (ADS)

Palkin, V. A.; Igoshin, I. S.

2017-01-01

The separation potentials suggested by various researchers for separating multicomponent isotopic mixtures are considered. An estimation of their applicability to determining the parameters of the efficiency of enrichment of a ternary mixture in a cascade with an optimum scheme of connection of stages made up of elements with three takeoffs is carried out. The separation potential most precisely characterizing the separative power and other efficiency parameters of stages and cascade schemes has been selected based on the results of the estimation made.

Pneumatic jigging: Influence of operating parameters on separation efficiency of solid waste materials.

PubMed

Abd Aziz, Mohd Aizudin; Md Isa, Khairuddin; Ab Rashid, Radzuwan

2017-06-01

This article aims to provide insights into the factors that contribute to the separation efficiency of solid particles. In this study, a pneumatic jigging technique was used to assess the separation of solid waste materials that consisted of copper, glass and rubber insulator. Several initial experiments were carried out to evaluate the strengths and limitations of the technique. It is found that despite some limitations of the technique, all the samples prepared for the experiments were successfully separated. The follow-up experiments were then carried out to further assess the separation of copper wire and rubber insulator. The effects of air flow and pulse rates on the separation process were examined. The data for these follow-up experiments were analysed using a sink float analysis technique. The analysis shows that the air flow rate was very important in determining the separation efficiency. However, the separation efficiency may be influenced by the type of materials used.

Fabrication and Wettability Study of WO3 Coated Photocatalytic Membrane for Oil-Water Separation: A Comparative Study with ZnO Coated Membrane.

PubMed

Gondal, Mohammed A; Sadullah, Muhammad S; Qahtan, Talal F; Dastageer, Mohamed A; Baig, Umair; McKinley, Gareth H

2017-05-10

Superhydrophilic and underwater superoleophobic surfaces were fabricated by facile spray coating of nanostructured WO 3 on stainless steel meshes and compared its performance in oil-water separation with ZnO coated meshes. The gravity driven oil-water separation system was designed using these surfaces as the separation media and it was noticed that WO 3 coated stainless steel mesh showed high separation efficiency (99%), with pore size as high as 150 µm, whereas ZnO coated surfaces failed in the process of oil-water separation when the pore exceeded 50 µm size. Since, nanostructured WO 3 is a well known catalyst, the simultaneous photocatalytic degradation of organic pollutants present in the separated water from the oil water separation process were tested using WO 3 coated surfaces under UV radiation and the efficiency of this degradation was found to be quite significant. These results assure that with little improvisation on the oil water separation system, these surfaces can be made multifunctional to work simultaneously for oil-water separation and demineralization of organic pollutants from the separated water. Fabrication of the separating surface, their morphological characteristics, wettability, oil water separation efficiency and photo-catalytic degradation efficiency are enunciated.

Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube.

PubMed

Kan, Hyo; Tsukagoshi, Kazuhiko

2017-07-01

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of Excipients on Liquid-Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions.

PubMed

Raut, Ashlesha S; Kalonia, Devendra S

2016-03-07

Liquid-liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature. In this study, the effect of four excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl beta-cyclodextrin (HPβCD)) on liquid-liquid phase separation and aggregation in a dual variable domain immunoglobulin protein solution was investigated. Sucrose suppressed both LLPS and aggregation, Tween 80 had no effect on either, and PEG 400 increased LLPS and aggregation. Attractive protein-protein interactions and liquid-liquid phase separation decreased with increasing concentration of HPβCD, indicating its specific binding to the protein. However, HPβCD had no effect on the formation of soluble aggregates and fragments in this study. LLPS and aggregation are highly temperature dependent; at low temperature protein exhibits LLPS, at high temperature protein exhibits aggregation, and at an intermediate temperature both phenomena occur simultaneously depending on the solution conditions.

Thinner, More-Efficient Oxygen-Separation Cells

NASA Technical Reports Server (NTRS)

Clark, Douglas J.; Galica, Leo M.; Losey, Robert W.

1992-01-01

Better gas-distribution plates fabricated more easily. Oxygen-separation cell redesigned to make it more efficient, smaller, lighter, and easier to manufacture. Potential applications include use as gas separators, filters, and fuel cells.

Ion-exchange chromatography purification of extracellular vesicles.

PubMed

Kosanović, Maja; Milutinović, Bojana; Goč, Sanja; Mitić, Ninoslav; Janković, Miroslava

2017-08-01

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

PubMed

Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

2017-01-20

This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass. Copyright © 2016 Elsevier B.V. All rights reserved.

Over 4,100 protein identifications from a Xenopus laevis fertilized egg digest using reversed-phase chromatographic prefractionation followed by capillary zone electrophoresis - electrospray ionization - tandem mass spectrometry analysis

PubMed Central

Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F.; Dovichi, Norman J.

2016-01-01

A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by reversed phase liquid chromatography. One set of 30 fractions was analyzed by 100-min CZE-ESI-MS/MS separations (50 hr total instrument time), and a second set of 15 fractions was analyzed by 3-hr UPLC-ESI-MS/MS separations (45 hr total instrument time). CZE-MS/MS produced 70% as many protein IDs (4,134 vs. 5,787) and 60% as many peptide IDs (22,535 vs. 36,848) as UPLC-MS/MS with similar instrument time (50 h vs. 45 h) but with 50 times smaller total consumed sample amount (1.5 μg vs. 75 μg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50-fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically-pumped nanospray interface used in CZE. This report is the first comparison of CZE-MS/MS and UPLC-MS/MS for large-scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE-ESI-MS/MS approach those produced by UPLC-MS/MS, but with nearly two orders of magnitude lower sample amounts. PMID:27723263

Novel Displacement Agents for Aqueous 2-Phase Extraction Can Be Estimated Based on Hybrid Shortcut Calculations.

PubMed

Kress, Christian; Sadowski, Gabriele; Brandenbusch, Christoph

2016-10-01

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate-polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients (B22 and B23) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B22 and experimentally validated within the citrate-PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Significance of surface charge and shell material of superparamagnetic iron oxide nanoparticle (SPION) based core/shell nanoparticles on the composition of the protein corona.

PubMed

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Coullerez, Geraldine; Hofmann-Amtenbrink, Margarethe; Vries, Marcel; Motazacker, Mahdi; Rezaee, Farhad; Hofmann, Heinrich

2015-02-01

As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) (positive, neutral and negative), SiO2 (positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas.

Protein separations using enhanced-fluidity liquid chromatography.

PubMed

Bennett, Raffeal; Olesik, Susan V

2017-11-10

Enhanced-fluidity liquid chromatography (EFLC) methods using methanol/H 2 O/CO 2 and hydrophilic interaction liquid chromatography (HILIC) were explored for the separation of proteins and peptides. EFLC is a separation mode that uses a mobile phase made of conventional solvents combined with liquid carbon dioxide (CO 2 ) in subcritical conditions. The addition of liquid CO 2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis. TFA additive and elevated temperature were leveraged as key factors in the separation of a 13-analyte intact protein mixture in under 5min. Under these conditions EFLC showed modest improvement in terms of peak asymmetry and analysis time over the competing ACN/H 2 O separation. Protein analytes detected by electrospray ionization - quadrupole time of flight, were shown to be unaffected by the addition of CO 2 in the mobile phase. Herein, the feasibility of separating hydrophilic proteins up to 80kDa (with transferrin) is demonstrated for CO 2 -containing mobile phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessing the scalability of dynamic field gradient focusing by linear modeling

PubMed Central

Tracy, Noah I.; Ivory, Cornelius F.

2010-01-01

Dynamic field gradient focusing (DFGF) separates and concentrates proteins in native buffers, where proteins are most soluble, using a computer-controlled electric field gradient which lets the operator adjust the pace and resolution of the separation in real-time. The work in this paper assessed whether DFGF could be scaled up from microgram analytical-scale protein loads to milligram preparative-scale loads. Linear modeling of the electric potential, protein transport, and heat transfer simulated the performance of a preparative-scale DFGF instrument. The electric potential model showed where the electrodes should be placed to optimize the shape and strength of the electric field gradient. Results from the protein transport model suggested that in 10 min the device should separate 10 mg each of two proteins whose electrophoretic mobilities differ by 5 ×. Proteins with electrophoretic mobilities differing by only 5% should separate in 3 h. The heat transfer model showed that the preparative DFGF design could dissipate 1 kW of Joule heat while keeping the separation chamber at 25°C. Model results pointed to DFGF successfully scaling up by 1000 × using the proposed instrument design. PMID:18196522

A Mixed Stimuli-Responsive Magnetic and Gold Nanoparticle System for Rapid Purification, Enrichment, and Detection of Biomarkers

PubMed Central

Nash, Michael A.; Yager, Paul; Hoffman, Allan S.; Stayton, Patrick S.

2010-01-01

A new diagnostic system for the enrichment and detection of protein biomarkers from human plasma is presented. Gold nanoparticles (AuNPs) were surface-modified with a diblock copolymer synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization. The diblock copolymer contained a thermally-responsive poly(N-isopropylacrylamide) (pNIPAAm) block, a cationic amine-containing block, and a semi-telechelic PEG2-biotin end group. When a mixed suspension of 23 nm pNIPAAm-modified AuNPs was heated with pNIPAAm-coated 10 nm iron oxide magnetic nanoparticles (mNPs) in human plasma, the thermally-responsive pNIPAAm directed the formation of mixed AuNP/mNP aggregates that could be separated efficiently with a magnet. Model studies showed that this mixed nanoparticle system could efficiently purify and strongly enrich the model biomarker protein streptavidin in spiked human plasma. A 10 ng/mL streptavidin sample was mixed with the biotinylated and pNIPAAm modified AuNP and magnetically separated in the mixed nanoparticle system with pNIPAAm mNPs. The aggregates were concentrated into a 50-fold smaller fluid volume at room temperature where the gold nanoparticle reagent redissolved with the streptavidin target still bound. The concentrated gold-labeled streptavidin could be subsequently analyzed directly using lateral flow immunochromatography. This rapid capture and enrichment module thus utilizes the mixed stimuli-responsive nanoparticle system to achieve direct concentration of a gold-labeled biomarker that can be directly analyzed using lateral flow or other rapid diagnostic strategies. PMID:21070026

Design of A Cyclone Separator Using Approximation Method

NASA Astrophysics Data System (ADS)

Sin, Bong-Su; Choi, Ji-Won; Lee, Kwon-Hee

2017-12-01

A Separator is a device installed in industrial applications to separate mixed objects. The separator of interest in this research is a cyclone type, which is used to separate a steam-brine mixture in a geothermal plant. The most important performance of the cyclone separator is the collection efficiency. The collection efficiency in this study is predicted by performing the CFD (Computational Fluid Dynamics) analysis. This research defines six shape design variables to maximize the collection efficiency. Thus, the collection efficiency is set up as the objective function in optimization process. Since the CFD analysis requires a lot of calculation time, it is impossible to obtain the optimal solution by linking the gradient-based optimization algorithm. Thus, two approximation methods are introduced to obtain an optimum design. In this process, an L18 orthogonal array is adopted as a DOE method, and kriging interpolation method is adopted to generate the metamodel for the collection efficiency. Based on the 18 analysis results, the relative importance of each variable to the collection efficiency is obtained through the ANOVA (analysis of variance). The final design is suggested considering the results obtained from two optimization methods. The fluid flow analysis of the cyclone separator is conducted by using the commercial CFD software, ANSYS-CFX.

Study of Anti-Fatigue Effect in Rats of Ferrous Chelates Including Hairtail Protein Hydrolysates

PubMed Central

Huang, Saibo; Lin, Huimin; Deng, Shang-gui

2015-01-01

The ability of ferrous chelates including hairtail protein hydrolysates to prevent and reduce fatigue was studied in rats. After hydrolysis of hairtail surimi with papain, the hairtail protein hydrolysates (HPH) were separated into three groups by range of relative molecular weight using ultrafiltration membrane separation. Hairtail proteins were then chelated with ferrous ions, and the antioxidant activity, the amino acid composition and chelation rate of the three kinds of ferrous chelates including hairtail protein hydrolysates (Fe-HPH) were determined. Among the three groups, the Fe-HPH chelate showing the best conditions was selected for the anti-fatigue animal experiment. For it, experimental rats were randomly divided into seven groups. Group A was designated as the negative control group given distilled water. Group B, the positive control group, was given glutathione. Groups C, D and E were designated as the Fe-HPH chelate treatment groups and given low, medium, and high doses, respectively. Group F was designated as HPH hydrolysate treatment group, and Group G was designated as FeCl2 treatment group. The different diets were orally administered to rats for 20 days. After that time, rats were subjected to forced swimming training after 1 h of gavage. Rats given Fe-FPH chelate had higher haemoglobin regeneration efficiency (HRE), longer exhaustive swimming time and higher SOD activity. Additionally, Fe-FPH chelate was found to significantly decrease the malondialdehyde content, visibly enhance the GSH-Px activity in liver and reduce blood lactic acid of rats. Fe-HPH chelate revealed an anti-fatigue effect, similar to or better than the positive control substance and superior to HPH or Fe when provided alone. PMID:26633476

Separation of proteins by hydrophobic interaction chromatography at low salt concentration.

PubMed

Kato, Yoshio; Nakamura, Koji; Kitamura, Takashi; Moriyama, Hiroyuki; Hasegawa, Masazumi; Sasaki, Hiroo

2002-09-20

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.

Position-dependent effects of polylysine on Sec protein transport.

PubMed

Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M

2012-04-13

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

Position-dependent Effects of Polylysine on Sec Protein Transport*

PubMed Central

Liang, Fu-Cheng; Bageshwar, Umesh K.; Musser, Siegfried M.

2012-01-01

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or “pause sites,” were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport. PMID:22367204

Chromatographic Studies of Protein-Based Chiral Separations

PubMed Central

Bi, Cong; Zheng, Xiwei; Azaria, Shiden; Beeram, Sandya; Li, Zhao; Hage, David S.

2016-01-01

The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha1-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations. PMID:28344977

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

Liu, Fang

Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Lyophilization, a practical way to store and transport tissues prior to protein extraction for 2DE analysis?

PubMed

Carpentier, Sebastien C; Dens, Koen; Van den houwe, Ines; Swennen, Rony; Panis, Bart

2007-09-01

To date, lyophilized samples are rarely utilized in proteomics experiments. This is most likely because researchers are concerned about inducing cross-linking of proteins via amide bonds, leading to artefactual charge modification and thus resulting in irreproducible results and bad gels. Indeed, it is known that lyophilization can cause crosslinking. The potential reaction is a reaction of free amino groups of a protein (N-terminal alpha-amino groups and epsilon-amino groups from lysine) with the reducing group of sugar molecules. The rate and extent of this reaction depends on the sugar content of the sample, the efficiency of lyophilization process, the residual water content of the material and the storage temperature. Lyophilization is a cheap, practical and safe alternative for the storage and transportation of samples prior to protein extraction, separation and quantification via 2DE, when care is taken (i) to dry the samples to the lowest practicable moisture content, (ii) to transport and store them under water- and airtight conditions and (iii) to avoid heating of the sample.

Fluorescent HPLC for the detection of specific protein oxidation carbonyls - α-aminoadipic and γ-glutamic semialdehydes - in meat systems.

PubMed

Utrera, Mariana; Morcuende, David; Rodríguez-Carpena, Javier-Germán; Estévez, Mario

2011-12-01

Precise methodologies for the routine analysis of particular protein carbonyls are required in order to progress in this topic of increasing interest. The present paper originally describes the application of an improved method for the detection of α-aminoadipic and γ-glutamic semialdehydes in a meat system by using a derivatization procedure with p-amino-benzoic acid (ABA) followed by fluorescent high-performance liquid chromatography (HPLC). The method development comprises i) the description of a simple HPLC program which allows the efficient separation of the ABA and the key standard compounds and ii) the optimization of the procedure for the preparation of a meat sample in order to maximize the fluorescent signal for both protein carbonyls. Furthermore, the suitability of this method is evaluated by applying the technique to porcine burger patties. The present procedure enables an accurate and relatively fast analysis of both semialdehydes in meat samples in which they could play an interesting role as reliable indicators of protein oxidation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots

PubMed Central

Tian, Li; Peel, Gregory J; Lei, Zhentian; Aziz, Naveed; Dai, Xinbin; He, Ji; Watson, Bonnie; Zhao, Patrick X; Sumner, Lloyd W; Dixon, Richard A

2009-01-01

Background White lupin (Lupinus albus L.) roots efficiently take up and accumulate (heavy) metals, adapt to phosphate deficiency by forming cluster roots, and secrete antimicrobial prenylated isoflavones during development. Genomic and proteomic approaches were applied to identify candidate genes and proteins involved in antimicrobial defense and (heavy) metal uptake and translocation. Results A cDNA library was constructed from roots of white lupin seedlings. Eight thousand clones were randomly sequenced and assembled into 2,455 unigenes, which were annotated based on homologous matches in the NCBInr protein database. A reference map of developing white lupin root proteins was established through 2-D gel electrophoresis and peptide mass fingerprinting. High quality peptide mass spectra were obtained for 170 proteins. Microsomal membrane proteins were separated by 1-D gel electrophoresis and identified by LC-MS/MS. A total of 74 proteins were putatively identified by the peptide mass fingerprinting and the LC-MS/MS methods. Genomic and proteomic analyses identified candidate genes and proteins encoding metal binding and/or transport proteins, transcription factors, ABC transporters and phenylpropanoid biosynthetic enzymes. Conclusion The combined EST and protein datasets will facilitate the understanding of white lupin's response to biotic and abiotic stresses and its utility for phytoremediation. The root ESTs provided 82 perfect simple sequence repeat (SSR) markers with potential utility in breeding white lupin for enhanced agronomic traits. PMID:19123941

Preservation of the soft protein corona in distinct flow allows identification of weakly bound proteins.

PubMed

Weber, C; Simon, J; Mailänder, V; Morsbach, S; Landfester, K

2018-06-08

Nanocarriers that are used for targeted drug delivery come in contact with biological liquids and subsequently proteins will adsorb to the nanocarriers' surface to form the so called 'protein corona'. The protein corona defines the biological identity and determines the biological response towards the nanocarriers in the body. To make nanomedicine safe and reliable it is required to get a better insight into this protein corona and, therefore, the adsorbed proteins have to be characterized. Currently, centrifugation is the common method to isolate the protein corona for further investigations. However, with this method it is only possible to investigate the strongly bound proteins, also referred to as 'hard protein corona'. Therefore, we want to introduce a new separation technique to separate nanoparticles including the soft protein corona containing also loosely bound proteins for further characterization. The used separation technique is the asymmetric flow field-flow fractionation (AF4). We were able to separate the nanoparticles with proteins forming the soft protein corona and were able to show that in our system only the hard protein corona directly influenced the cell uptake behavior. Currently, there is an ongoing debate whether only strongly bound proteins (hard corona) or also loosely bound proteins (soft corona) contribute to the biological identity of nanocarriers, because up to now isolation of the soft corona was not possible. Here, asymmetric flow field-flow fractionation was used to isolate nanoparticles with a preserved soft corona from the biological medium. This enabled the characterization of the soft corona composition and to evaluate its influence on cellular uptake. For our system we found that only the strongly bound proteins (hard corona) determined cell internalization. This method can now be used to evaluate the impact of the soft corona further and to characterize nanomaterials that cannot be separated from blood plasma by other means. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Membraneless laminar flow cell for electrocatalytic CO2 reduction with liquid product separation

NASA Astrophysics Data System (ADS)

Monroe, Morgan M.; Lobaccaro, Peter; Lum, Yanwei; Ager, Joel W.

2017-04-01

The production of liquid fuel products via electrochemical reduction of CO2 is a potential path to produce sustainable fuels. However, to be practical, a separation strategy is required to isolate the fuel-containing electrolyte produced at the cathode from the anode and also prevent the oxidation products (i.e. O2) from reaching the cathode. Ion-conducting membranes have been applied in CO2 reduction reactors to achieve this separation, but they represent an efficiency loss and can be permeable to some product species. An alternative membraneless approach is developed here to maintain product separation through the use of a laminar flow cell. Computational modelling shows that near-unity separation efficiencies are possible at current densities achievable now with metal cathodes via optimization of the spacing between the electrodes and the electrolyte flow rate. Laminar flow reactor prototypes were fabricated with a range of channel widths by 3D printing. CO2 reduction to formic acid on Sn electrodes was used as the liquid product forming reaction, and the separation efficiency for the dissolved product was evaluated with high performance liquid chromatography. Trends in product separation efficiency with channel width and flow rate were in qualitative agreement with the model, but the separation efficiency was lower, with a maximum value of 90% achieved.

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

PubMed

Ding, Xueqin; Wang, Yuzhi; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

2014-03-07

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly Efficient Quantum Sieving in Porous Graphene-like Carbon Nitride for Light Isotopes Separation

NASA Astrophysics Data System (ADS)

Qu, Yuanyuan; Li, Feng; Zhou, Hongcai; Zhao, Mingwen

2016-01-01

Light isotopes separation, such as 3He/4He, H2/D2, H2/T2, etc., is crucial for various advanced technologies including isotope labeling, nuclear weapons, cryogenics and power generation. However, their nearly identical chemical properties made the separation challenging. The low productivity of the present isotopes separation approaches hinders the relevant applications. An efficient membrane with high performance for isotopes separation is quite appealing. Based on first-principles calculations, we theoretically demonstrated that highly efficient light isotopes separation, such as 3He/4He, can be reached in a porous graphene-like carbon nitride material via quantum sieving effect. Under moderate tensile strain, the quantum sieving of the carbon nitride membrane can be effectively tuned in a continuous way, leading to a temperature window with high 3He/4He selectivity and permeance acceptable for efficient isotopes harvest in industrial application. This mechanism also holds for separation of other light isotopes, such as H2/D2, H2/T2. Such tunable quantum sieving opens a promising avenue for light isotopes separation for industrial application.

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

Wu, Chaochao; Duan, Jicheng; Liu, Tao

Human biofluids, especially blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concentrations in biofluids represents a significant analytical challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatographic methods have been developed and routinely applied for separating low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity separation methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity separation methodsmore » are also discussed.« less

Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis.

PubMed

Li, Ping; Shao, Yize; Jin, Hui; Yu, Hong-Guo

2015-04-27

Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. © 2015 Li et al.

Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

PubMed Central

Li, Ping; Shao, Yize; Jin, Hui

2015-01-01

Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

PubMed Central

Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

2012-01-01

In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

PubMed Central

Boudreau, Amy C.; Milovanovic, Mike; Conrad, Kelly L.; Nelson, Christopher; Ferrario, Carrie R.; Wolf, Marina E.

2012-01-01

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then cell surface-expressed receptors are covalently crosslinked to nearby proteins using the membrane-impermeable, bifunctional crosslinker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after crosslinking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. PMID:22470150

Halloysite-based dopamine-imprinted polymer for selective protein capture.

PubMed

Zhu, Xiaohong; Li, Hui; Liu, Hui; Peng, Wei; Zhong, Shian; Wang, Yan

2016-06-01

We describe a facile, general, and highly efficient approach to obtain polydopamine-coated molecularly imprinted polymer based on halloysite nanotubes for bovine serum albumin. The method combined surface molecular imprinting and one-step immobilized template technique. Hierarchically structured polymer was prepared in physiological conditions adopting dopamine as functional monomer. A thin layer of polydopamine can be coated on the surface of amino-modified halloysite nanotubes by self-polymerization, and the thickness of the imprinted shells can be controlled by the mass ratio of matrix and dopamine. The polymer was characterized by Fourier transform infrared spectrometry, transmission electron microscopy, and thermogravimetric analysis. The prepared material showed high binding capacity (45.4 mg/g) and specific recognition behavior toward the template protein. In addition, stability and regeneration analyses indicated that the imprinted polymer exhibited excellent reusability (relative standard deviation < 9% for batch-to-batch evaluation). Therefore, the developed polymer is effective for protein recognition and separation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Environmental friendly cold-mechanical/sonic enzymatic assisted extraction of genipin from genipap (Genipa americana).

PubMed

Ramos-de-la-Peña, Ana Mayela; Renard, Catherine M G C; Wicker, Louise; Montañez, Julio C; García-Cerda, Luis Alfonso; Contreras-Esquivel, Juan Carlos

2014-01-01

An efficient cold-mechanical/sonic-assisted extraction technique was developed for extraction of genipin from genipap (Genipa americana) peel. Ultrasound assisted extraction (285 W, 24 kHz) was performed at 5, 10 and 15 °C for 5, 10 and 15 min. After cold-extraction, genipin was separated from pectin and proteins by aid of fungal pectinesterase. The maximum yield of non-cross-linked genipin was 7.85±0.33 mg/g, at 10 °C for 15 min by means of ultrasound extraction. The protein amount in extracts decreased in all samples. If mechanical process is combined with ultrasound assisted extraction the yield is increased by 8 times after the pectinesterase-assisted polyelectrolyte complex formation between pectic polysaccharides and proteins, avoiding the typical cross-linking of genipin. This novel process is viable to obtain non-cross-linked genipin, to be used as a natural colorant and cross-linker in the food and biotechnological industries. Copyright © 2013 Elsevier B.V. All rights reserved.

Automatic poisson peak harvesting for high throughput protein identification.

PubMed

Breen, E J; Hopwood, F G; Williams, K L; Wilkins, M R

2000-06-01

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

Controlling Gas-Phase Reactions for Efficient Charge Reduction Electrospray Mass Spectrometry of Intact Proteins

PubMed Central

Frey, Brian L.; Lin, Yuan; Westphall, Michael S.; Smith, Lloyd M.

2006-01-01

Charge reduction electrospray mass spectrometry (CREMS) reduces the charge states of electrospray-generated ions, which concentrates the ions from a protein into fewer peaks spread over a larger m/z range, thereby increasing peak separation and decreasing spectral congestion. An optimized design for a CREMS source is described that provides an order-of-magnitude increase in sensitivity compared to previous designs and provides control over the extent of charge reduction. Either a corona discharge or an α-particle source was employed to generate anions that abstract protons from electrosprayed protein cations. These desired ion/ion proton transfer reactions predominated, but some oxidation and ion-attachment reactions also occurred leading to new peaks or mass-shifted broader peaks while decreasing signal intensity. The species producing these deleterious side-reactions were identified, and conditions were found that prevented their formation. Spectrometer m/z biases were examined because of their effect upon the signal intensity of higher m/z charge-reduced protein ions. The utility of this atmospheric pressure CREMS was demonstrated using a cell lysate fraction from E. coli. The spectral simplification afforded by CREMS reveals more proteins than are observed without charge reduction. PMID:16198118

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro.

PubMed

Todd, Christopher D; Gifford, David J

2002-05-01

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Endoplasmic reticulum mediated signaling in cellular microdomains

PubMed Central

Biwer, Lauren; Isakson, Brant E

2016-01-01

The endoplasmic reticulum (ER) is a prime mediator of cellular signaling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitate calcium and lipid signaling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability in order to project into restricted spaces. The signaling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signaling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER-mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+]i, and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signaling, which is important for cellular physiology and organism homeostasis. PMID:26973141

Microfluidic size separation of cells and particles using a swinging bucket centrifuge.

PubMed

Yeo, Joo Chuan; Wang, Zhiping; Lim, Chwee Teck

2015-09-01

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.

Microfluidic size separation of cells and particles using a swinging bucket centrifuge

PubMed Central

Yeo, Joo Chuan; Wang, Zhiping; Lim, Chwee Teck

2015-01-01

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency. PMID:26487900

Efficient 3He/4He separation in a nanoporous graphenylene membrane.

PubMed

Qu, Yuanyuan; Li, Feng; Zhao, Mingwen

2017-08-16

Helium-3 is a precious noble gas, which is essential in many advanced technologies such as cryogenics, isotope labeling and nuclear weapons. The current imbalance of 3 He demand and supply shortage leads to the search for an efficient membrane with high performance for 3 He separation. In this study, based on first-principles calculations, we demonstrated that highly efficient 3 He harvesting can be achieved in a nanoporous graphenylene membrane with industrially-acceptable selectivity and permeance. The quantum tunneling effect leads to 3 He harvesting with high efficiency via kinetic sieving. Both the quantum tunneling effect and zero-point energy (ZPE) determine the 3 He/ 4 He separation via thermally-driven equilibrium sieving, where the ZPE effect dominates efficient 3 He/ 4 He separation between two reservoirs. The quantum effects revealed in this work suggest that the nanoporous graphenylene membrane is promising for efficient 3 He harvesting that can be exploited for industrial applications.

Droplet-based magnetically activated cell separation: analysis of separation efficiency based on the variation of flow-induced circulation in a pendent drop.

PubMed

Kim, Youngho; Lee, Sang Ho; Kim, Byungkyu

2009-12-01

Under the assumption that separation efficiencies are mainly affected by the velocity of flow-induced circulation due to buffer injection in a pendent drop, this paper describes an analysis of the separation efficiency of a droplet-based magnetically activated cell separation (DMACS) system. To investigate the velocity of the flow-induced circulation, we supposed that numerous flows in a pendent drop could be considered as a "theoretically normalized" flow (or conceptually normalized flow, CNF) based on the Cauchy-Goursat theorem. With the morphological characteristics (length and duration time) of a pendent drop depending on the initial volume, we obtained the velocities of the CNF. By measuring the separation efficiencies for different initial volumes and by analyzing the separation efficiency in terms of the velocity of the CNF, we found that the separation efficiencies (in the case of a low rate of buffer injection; 5 and 15 microl x min(-1)) are mainly affected by the velocity of the CNF. Moreover, we confirmed that the phenomenological features of a pendent drop cause a fluctuation of its separation efficiencies over a range of specific volumes (initial volumes ranging from 40 to 80 microl), because of the "sweeping-off" phenomenon, that is, positive cells gathered into the positive fraction are forced to move away from the magnetic side by flow-induced circulation due to buffer injection. In addition, from the variation of the duration time, that is, the interval between the beginning of injection of the buffer solution and the time at which a pendent drop detaches, it could also be confirmed that a shorter duration time leads to decrease of the number of positive cells in negative fraction regardless of the rate of buffer injection (5, 15, and 50 microl x min(-1)). Therefore, if a DMACS system is operated with a 15 microl x min(-1) buffer injection flow rate and an initial volume of 80 microl or more, we would have the best efficiency of separation in the negative fraction.

Separation of patatins and protease inhibitors from potato fruit juice with clay minerals as cation exchangers.

PubMed

Ralla, Kathrin; Sohling, Ulrich; Suck, Kirstin; Kasper, Cornelia; Ruf, Friedrich; Scheper, Thomas

2012-07-01

Potato fruit juice as a by-product of the starch industry contains proteins with interesting functionalities such as protease inhibitors or patatin with its high nutritional value. Due to their functional properties, these proteins are principally of industrial interest. A drawback for the application of these potato proteins is the separation and isolation under maintenance of the biological activity. So far, there are no methods in literature, which are satisfying concerning the costs or the separation performance. In this study, we show a chromatographic approach using natural clay minerals as cation exchangers to separate two protein fractions in potato fruit juice. Additionally, the content of glycoalkaloids naturally occurring in potatoes is significantly reduced in a single step together with the separation of the patatins and the protease inhibitors. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous and selective decarboxylation of L-serine and deamination of L-phenylalanine in an amino acid mixture--a means of separating amino acids for synthesizing biobased chemicals.

PubMed

Teng, Yinglai; Scott, Elinor L; Witte-van Dijk, Susan C M; Sanders, Johan P M

2016-01-25

Amino acids (AAs) obtained from the hydrolysis of biomass-derived proteins are interesting feedstocks for the chemical industry. They can be prepared from the byproduct of biofuel production and agricultural wastes. They are rich in functionalities needed in petrochemicals, providing the opportunity to save energy, reagents, and process steps. However, their separation is required before they can be applied for further applications. Electrodialysis (ED) is a promising separation method, but its efficiency needs to be improved when separating AAs with similar isoelectric points. Thus, specific conversions are required to form product with different charges. Here we studied the enzymatic conversions which can be used as a means to aid the ED separation of neutral AAs. A model mixture containing L-serine, L-phenylalanine and L-methionine was used. The reactions of L-serine decarboxylase and L-phenylalanine ammonia-lyase were employed to specifically convert serine and phenylalanine into ethanolamine and trans-cinnamic acid. At the isoelectric point of methionine (pH 5.74), the charge of ethanolamine and trans-cinnamic acid are +1 and -1, therefore facilitating potential separation into three different streams by electrodialysis. Here the enzyme kinetics, specificity, inhibition and the operational stabilities were studied, showing that both enzymes can be applied simultaneously to aid the ED separation of neutral AAs. Copyright © 2015 Elsevier B.V. All rights reserved.

Photosynthetic Machineries in Nano-Systems

PubMed Central

Nagy, László; Magyar, Melinda; Szabó, Tibor; Hajdu, Kata; Giotta, Livia; Dorogi, Márta; Milano, Francesco

2014-01-01

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field. PMID:24678673

RGD and polyhistidine tumor homing peptides potentiates the action of human Maspin as an antineoplastic candidate.

PubMed

Yin, Runting; Guo, Le; Zhang, Jie; Liu, Guangzhao; Yao, Wenjuan; Zhu, Hongyan; Xu, Xiaole; Zhang, Wei

2016-07-01

Maspin, a non-inhibitory member of serine protease family, acts as an effective tumor suppressor by inhibiting cell inhesion and mobility. We found that exogenous wild-type rMaspin had a low effect on tumor growth in vivo. However, when the peptide Arg-Gly-Asp-hexahistidine (RGD-6His) was introduced into rMaspin, the modified rMaspin showed significant inhibitory activity in angiogenic assays and tumor-bearing animal models. Overall, our data suggested that both the RGD and hexahistidine fragments contributed to improve the fusion protein activity and polyhistidine peptide could be considered as flexible linker to separate RGD and Maspin moieties to avoid function interference. Besides, it is an efficient tag to achieve purified recombinant proteins. Furthermore, rMaspin fusing with RGD and hexahistidine could be a viable anticancer candidate.

Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation.

PubMed

Cheng, Ya-Ling; Juang, Yu-Chuan; Liao, Guan-Yu; Tsai, Pei-Wen; Ho, Shih-Hsin; Yeh, Kuei-Ling; Chen, Chun-Yen; Chang, Jo-Shu; Liu, Jhy-Chern; Chen, Wen-Ming; Lee, Duu-Jong

2011-01-01

The Scenedesmus obliquus FSP-3, a species with excellent potential for CO(2) capture and lipid production, was harvested using dispersed ozone flotation. While air aeration does not, ozone produces effective solid-liquid separation through flotation. Ozone dose applied for sufficient algal flotation is similar to those used in practical drinking waterworks. The algae removal rate, surface charge, and hydrophobicity of algal cells, and fluorescence characteristics and proteins and polysaccharides contents of algogenic organic matter (AOM) were determined during ozonation. Proteins released from tightly bound AOM are essential to modifying the hydrophobicity of bubble surfaces for easy cell attachment and to forming a top froth layer for collecting floating cells. Humic substances in the suspension scavenge dosed ozone that adversely affects ozone flotation efficiency of algal cells. Copyright © 2010 Elsevier Ltd. All rights reserved.

Photoactive films of photosystem I on transparent reduced graphene oxide electrodes.

PubMed

Darby, Emily; LeBlanc, Gabriel; Gizzie, Evan A; Winter, Kevin M; Jennings, G Kane; Cliffel, David E

2014-07-29

Photosystem I (PSI) is a photoactive electron-transport protein found in plants that participates in the process of photosynthesis. Because of PSI's abundance in nature and its efficiency with charge transfer and separation, there is a great interest in applying the protein in photoactive electrodes. Here, we developed a completely organic, transparent, conductive electrode using reduced graphene oxide (RGO) on which a multilayer of PSI could be deposited. The resulting photoactive electrode demonstrated current densities comparable to that of a gold electrode modified with a multilayer film of PSI and significantly higher than that of a graphene electrode modified with a monolayer film of PSI. The relatively large photocurrents produced by integrating PSI with RGO and using an opaque, organic mediator can be applied to the facile production of more economic solar energy conversion devices.

A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

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

Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl

2009-10-02

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this systemmore » using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.« less

Metadynamic metainference: Enhanced sampling of the metainference ensemble using metadynamics

PubMed Central

Bonomi, Massimiliano; Camilloni, Carlo; Vendruscolo, Michele

2016-01-01

Accurate and precise structural ensembles of proteins and macromolecular complexes can be obtained with metainference, a recently proposed Bayesian inference method that integrates experimental information with prior knowledge and deals with all sources of errors in the data as well as with sample heterogeneity. The study of complex macromolecular systems, however, requires an extensive conformational sampling, which represents a separate challenge. To address such challenge and to exhaustively and efficiently generate structural ensembles we combine metainference with metadynamics and illustrate its application to the calculation of the free energy landscape of the alanine dipeptide. PMID:27561930

Efficient separation of conjugated polymers using a water soluble glycoprotein matrix: from fluorescence materials to light emitting devices.

PubMed

Hendler, Netta; Wildeman, Jurjen; Mentovich, Elad D; Schnitzler, Tobias; Belgorodsky, Bogdan; Prusty, Deepak K; Rimmerman, Dolev; Herrmann, Andreas; Richter, Shachar

2014-03-01

Optically active bio-composite blends of conjugated polymers or oligomers are fabricated by complexing them with bovine submaxilliary mucin (BSM) protein. The BSM matrix is exploited to host hydrophobic extended conjugated π-systems and to prevent undesirable aggregation and render such materials water soluble. This method allows tuning the emission color of solutions and films from the basic colors to the technologically challenging white emission. Furthermore, electrically driven light emitting biological devices are prepared and operated. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CE-microreactor-CE-MS/MS for protein analysis

PubMed Central

Schoenherr, Regine M.; Ye, Mingliang; Vannatta, Michael

2008-01-01

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome C and myoglobin generated Mascot MOWSE scores of 107 for cytochrome C and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively. PMID:17295444

Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography

PubMed Central

Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei

2016-01-01

Microfluidic chip electrophoresis (MCE) is a powerful separation tool for biomacromolecule analysis. However, adsorption of biomacromolecules, particularly proteins onto microfluidic channels severely degrades the separation performance of MCE. In this paper, an anti-protein-fouling MCE was fabricated using a novel sandwich photolithography of poly(ethylene glycol) (PEG) prepolymers. Photopatterned microchannel with a minimum resolution of 10 μm was achieved. After equipped with a conventional online electrochemical detector, the device enabled baseline separation of bovine serum albumin, lysozyme (Lys), and cytochrome c (Cyt-c) in 53 s under a voltage of 200 V. Compared with a traditional polydimethylsiloxane MCE made by soft lithography, the PEG MCE made by the sandwich photolithography not only eliminated the need of a master mold and the additional modification process of the microchannel but also showed excellent anti-protein-fouling properties for protein separation. PMID:27493702

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

PubMed

Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison E; Han, Jongyoon; Alter, Galit

2016-03-30

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

Spectral unmixing of multi-color tissue specific in vivo fluorescence in mice

NASA Astrophysics Data System (ADS)

Zacharakis, Giannis; Favicchio, Rosy; Garofalakis, Anikitos; Psycharakis, Stylianos; Mamalaki, Clio; Ripoll, Jorge

2007-07-01

Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo. The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for specific applications.

Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

PubMed Central

Foda, Bardees M.; Downey, Kurtis M.; Fisk, John C.

2012-01-01

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

Energetics of bacterial photosynthesis.

PubMed

Lebard, David N; Matyushov, Dmitry V

2009-09-10

We report the results of extensive numerical simulations and theoretical calculations of electronic transitions in the reaction center of Rhodobacter sphaeroides photosynthetic bacterium. The energetics and kinetics of five electronic transitions related to the kinetic scheme of primary charge separation have been analyzed and compared to experimental observations. Nonergodic formulation of the reaction kinetics is required for the calculation of the rates due to a severe breakdown of the system ergodicity on the time scale of primary charge separation, with the consequent inapplicability of the standard canonical prescription to calculate the activation barrier. Common to all reactions studied is a significant excess of the charge-transfer reorganization energy from the width of the energy gap fluctuations over that from the Stokes shift of the transition. This property of the hydrated proteins, breaking the linear response of the thermal bath, allows the reaction center to significantly reduce the reaction free energy of near-activationless electron hops and thus raise the overall energetic efficiency of the biological charge-transfer chain. The increase of the rate of primary charge separation with cooling is explained in terms of the temperature variation of induction solvation, which dominates the average donor-acceptor energy gap for all electronic transitions in the reaction center. It is also suggested that the experimentally observed break in the Arrhenius slope of the primary recombination rate, occurring near the temperature of the dynamical transition in proteins, can be traced back to a significant drop of the solvent reorganization energy close to that temperature.

Binding of [51Cr]ethylenediaminetetraacetate to proteins of human plasma.

PubMed Central

Babiker, M M

1986-01-01

Binding of [51Cr]EDTA to human plasma proteins was investigated using chemical and chromatographic techniques of separation of the proteins and protein fractions. Total plasma proteins isolated with ethanol retained 12.95 +/- 0.46% of the initial plasma activity. Proteins separated by other precipitants retained about 16% of the initial radioactivity. Globulins exhibited the highest binding capacity for [51Cr]EDTA and retained about 11.7% of the initial plasma activity following chromatographic separation. This value represents about 70% of the radioactivity bound by the total proteins of the plasma. gamma-Globulins contributed most of the binding attributed to the globulins and retained about 8.7% of the initial [51Cr]EDTA activity. The repeatedly reported underestimation of the renal glomerular filtration rate when estimated as the clearance of [51Cr]EDTA could be adequately accounted for by the extent of binding of this marker to the plasma proteins. PMID:2427701

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

PubMed

Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

2005-09-01

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.

Efficient hydrogen isotopologues separation through a tunable potential barrier: The case of a C2N membrane.

PubMed

Qu, Yuanyuan; Li, Feng; Zhao, Mingwen

2017-05-03

Isotopes separation through quantum sieving effect of membranes is quite promising for industrial applications. For the light hydrogen isotopologues (eg. H 2 , D 2 ), the confinement of potential wells in porous membranes to isotopologues was commonly regarded to be crucial for highly efficient separation ability. Here, we demonstrate from first-principles that a potential barrier is also favorable for efficient hydrogen isotopologues separation. Taking an already-synthesized two-dimensional carbon nitride (C 2 N-h2D) as an example, we predict that the competition between quantum tunneling and zero-point-energy (ZPE) effects regulated by the tensile strain leads to high selectivity and permeance. Both kinetic quantum sieving and equilibrium quantum sieving effects are considered. The quantum effects revealed in this work offer a prospective strategy for highly efficient hydrogen isotopologues separation.

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465].

PubMed

Tsymbal, N V; Samoĭlenko, V A; Syrchin, S A; Mendzhul, M I

2004-01-01

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

The microbial transglutaminase immobilization on carboxylated poly(N-isopropylacrylamide) for thermo-responsivity.

PubMed

Zhou, Jian Qin; He, Ting; Wang, Jian Wen

2016-06-01

Microbial transglutaminase (mTG) is widely utilized in the PEGylation of pharmaceutical proteins. mTG immobilization can be achieved via covalent bonding on solid supports. However, the catalytic efficiency of mTG immobilized on solid supports was significantly reduced by mass transfer limitation. To overcome this limitation, mTG was covalently immobilized on the thermo-responsive carboxylated poly(N-isopropylacrylamide) (pNIPAM). The pNIPAM-mTG conjugate exhibited reversibly solubility in aqueous solution with a low critical solution temperature (LCST) at 39°C, i.e., it was insoluble above 39°C and soluble below 39°C. The pH dependence of the pNIPAM-mTG conjugate was similar with that of the native mTG. Upon conjugation to pNIPAM, the optimal temperature of mTG shifted down from 50-55°C to 40-45°C, and the thermal stability of the conjugate was elevated. The easy separation of the pNIPAM-mTG conjugate with its substrate and the catalytic efficiency of the pNIPAM-mTG conjugate were demonstrated by employing the pNIPAM-mTG conjugate to cross-link bovine serum albumin (BSA) and catalyze PEGylation of therapeutic protein, cytochrome c (Cyt C), respectively. The thermo-responsive mTG is suitable to modify proteins in food processing and biomedical engineering. Copyright © 2016 Elsevier Inc. All rights reserved.

Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids

DOE PAGES

Wu, Chaochao; Duan, Jicheng; Liu, Tao; ...

2016-01-12

Human biofluids, especially blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concentrations in biofluids represents a significant analytical challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatographic methods have been developed and routinely applied for separating low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity separation methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity separation methodsmore » are also discussed.« less

Binary Oscillatory Crossflow Electrophoresis

NASA Technical Reports Server (NTRS)

Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

1997-01-01

Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that solute is drawn into the cell from reservoirs at both ends of the cell leading to a large mass build up. As a consequence, any initially induced mass flux will vanish after short times. This effect was not captured by the infinite channel model and hence numerical and experimental results deviated significantly. The revised model including finite cell lengths and reservoir volumes allowed quantitative predictions of the time history of the concentration profile throughout the system. This latter model accurately describes the fluxes observed for both oscillatory flow modes in experiments using single protein species. Based on the results obtained from research funded under NASA grant NAG-8-1080.S, we conclude that binary separations are not possible using purely oscillatory flow modes because of end effects associated with the cos((omega)t) mode. Our research shows, however, that a combination of cos(2(omega)t) and steady flow should lead to efficient separation free of end effects. This possibility is currently under investigation.

Membraneless laminar flow cell for electrocatalytic CO 2 reduction with liquid product separation

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

Monroe, Morgan M.; Lobaccaro, Peter; Lum, Yanwei

The production of liquid fuel products via electrochemical reduction of CO 2 is a potential path to produce sustainable fuels. However, to be practical, a separation strategy is required to isolate the fuel-containing electrolyte produced at the cathode from the anode and also prevent the oxidation products (i.e. O 2) from reaching the cathode. Ion-conducting membranes have been applied in CO 2 reduction reactors to achieve this separation, but they represent an efficiency loss and can be permeable to some product species. An alternative membraneless approach is developed here to maintain product separation through the use of a laminar flowmore » cell. Computational modelling shows that near-unity separation efficiencies are possible at current densities achievable now with metal cathodes via optimization of the spacing between the electrodes and the electrolyte flow rate. Laminar flow reactor prototypes were fabricated with a range of channel widths by 3D printing. CO 2 reduction to formic acid on Sn electrodes was used as the liquid product forming reaction, and the separation efficiency for the dissolved product was evaluated with high performance liquid chromatography. Trends in product separation efficiency with channel width and flow rate were in qualitative agreement with the model, but the separation efficiency was lower, with a maximum value of 90% achieved.« less

Membraneless laminar flow cell for electrocatalytic CO 2 reduction with liquid product separation

DOE PAGES

Monroe, Morgan M.; Lobaccaro, Peter; Lum, Yanwei; ...

2017-03-16

The production of liquid fuel products via electrochemical reduction of CO 2 is a potential path to produce sustainable fuels. However, to be practical, a separation strategy is required to isolate the fuel-containing electrolyte produced at the cathode from the anode and also prevent the oxidation products (i.e. O 2) from reaching the cathode. Ion-conducting membranes have been applied in CO 2 reduction reactors to achieve this separation, but they represent an efficiency loss and can be permeable to some product species. An alternative membraneless approach is developed here to maintain product separation through the use of a laminar flowmore » cell. Computational modelling shows that near-unity separation efficiencies are possible at current densities achievable now with metal cathodes via optimization of the spacing between the electrodes and the electrolyte flow rate. Laminar flow reactor prototypes were fabricated with a range of channel widths by 3D printing. CO 2 reduction to formic acid on Sn electrodes was used as the liquid product forming reaction, and the separation efficiency for the dissolved product was evaluated with high performance liquid chromatography. Trends in product separation efficiency with channel width and flow rate were in qualitative agreement with the model, but the separation efficiency was lower, with a maximum value of 90% achieved.« less

Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm

NASA Astrophysics Data System (ADS)

Austin, Robert; Wunsch, Benjamin; Smith, Joshua; Gifford, Stacey; Wang, Chao; Brink, Markus; Bruce, Robert; Stolovitzky, Gustavo; Astier, Yann

Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites1, bacteria2, blood cells3 and circulating tumour cells in blood4. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of liquid biopsies, are secreted by cells and contain nucleic acid and protein information about their originating tissue5. One challenge in the study of exosome biology is to sort exosomes by size and surface markers6, 7. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235 nm. We show that at low Péclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110 nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm

NASA Astrophysics Data System (ADS)

Wunsch, Benjamin H.; Smith, Joshua T.; Gifford, Stacey M.; Wang, Chao; Brink, Markus; Bruce, Robert L.; Austin, Robert H.; Stolovitzky, Gustavo; Astier, Yann

2016-11-01

Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites, bacteria, blood cells and circulating tumour cells in blood. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of 'liquid biopsies', are secreted by cells and contain nucleic acid and protein information about their originating tissue. One challenge in the study of exosome biology is to sort exosomes by size and surface markers. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235 nm. We show that at low Péclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110 nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

Highly efficient peptide separations in proteomics Part 1. Unidimensional high performance liquid chromatography.

PubMed

Sandra, Koen; Moshir, Mahan; D'hondt, Filip; Verleysen, Katleen; Kas, Koen; Sandra, Pat

2008-04-15

Sample complexity and dynamic range constitute enormous challenges in proteome analysis. The back-end technology in typical proteomics platforms, namely mass spectrometry (MS), can only tolerate a certain complexity, has a limited dynamic range per spectrum and is very sensitive towards ion suppression. Therefore, component overlap has to be minimized for successful mass spectrometric analysis and subsequent protein identification and quantification. The present review describes the advances that have been made in liquid-based separation techniques with focus on the recent developments to boost the resolving power. The review is divided in two parts; the first part deals with unidimensional liquid chromatography and the second part with bi- and multidimensional liquid-based separation techniques. Part 1 mainly focuses on reversed-phase HPLC due to the fact that it is and will, in the near future, remain the technique of choice to be hyphenated with MS. The impact of increasing the column length, decreasing the particle diameter, replacing the traditional packed beds by monolithics, amongst others, is described. The review is complemented with data obtained in the laboratories of the authors.

A superhydrophobic copper mesh as an advanced platform for oil-water separation

NASA Astrophysics Data System (ADS)

Ren, Guina; Song, Yuanming; Li, Xiangming; Zhou, Yanli; Zhang, Zhaozhu; Zhu, Xiaotao

2018-01-01

Improving the separation efficiency and simplifying the separation process would be highly desired for oil-water separation yet still challenging. Herein, to address this challenge, we fabricated a superhydrophobic copper mesh by an immersion process and exploited it as an advanced platform for oil-water separation. To realize oil-water separation efficiently, the obtained mesh was enfolded directly to form a boat-like device, and it could also be mounted on an open end of a glass barrel to form the oil skimmer device. For these devices, they can collect the floating oils through the pores of the copper mesh while repelling water completely, and the oil collection efficiency is up to 99.5%. Oils collected in the devices can be easily sucked out into a container for storing, without requiring mechanical handing for recycling. Importantly, the miniature boat and the oil skimmer devices can retain their enhanced oil collection efficiency even after 10 cycles of oil-water separation. Moreover, exploiting its superhydrophobicity under oil, the obtained copper mesh was demonstrated as a novel platform to remove tiny water droplets from oil.

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

PubMed

Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

2012-08-30

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid chromatography with a single column (2DLC-1C), which can also be employed to separate three kinds of active proteins completely, such as lysozyme, ovotransferrin and ovalbumin from egg white. The result is very important not only to the development of new 2DLC technology with a single column for proteomics, but also to recombinant protein drug production for saving column expense and simplifying the process in biotechnology. Copyright © 2012 Elsevier B.V. All rights reserved.

Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

PubMed

Bengali, Aditya N; Tessier, Peter M

2009-10-01

"Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these interactions. (c) 2009 Wiley Periodicals, Inc.

Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatography-mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides.

PubMed

Eeltink, Sebastiaan; Wouters, Sam; Dores-Sousa, José Luís; Svec, Frantisek

2017-05-19

This review focuses on the preparation of organic polymer-based monolithic stationary phases and their application in the separation of biomolecules, including antibodies, intact proteins and protein isoforms, oligonucleotides, and protein digests. Column and material properties, and the optimization of the macropore structure towards kinetic performance are also discussed. State-of-the-art liquid chromatography-mass spectrometry biomolecule separations are reviewed and practical aspects such as ion-pairing agent selection and carryover are presented. Finally, advances in comprehensive two-dimensional LC separations using monolithic columns, in particular ion-exchange×reversed-phase and reversed-phase×reversed-phase LC separations conducted at high and low pH, are shown. Copyright © 2017 Elsevier B.V. All rights reserved.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion

PubMed Central

Sol, E-ri M; Hovsepyan, Meri; Bergsten, Peter

2009-01-01

Background Development of type 2 diabetes mellitus (T2DM) is characterized by aberrant insulin secretory patterns, where elevated insulin levels at non-stimulatory basal conditions and reduced hormonal levels at stimulatory conditions are major components. To delineate mechanisms responsible for these alterations we cultured INS-1E cells for 48 hours at 20 mM glucose in absence or presence of 0.5 mM palmitate, when stimulatory secretion of insulin was reduced or basal secretion was elevated, respectively. Results After culture, cells were protein profiled by SELDI-TOF-MS and 2D-PAGE. Differentially expressed proteins were discovered and identified by peptide mass fingerprinting. Complimentary protein profiles were obtained by the two approaches with SELDI-TOF-MS being more efficient in separating proteins in the low molecular range and 2D-PAGE in the high molecular range. Identified proteins included alpha glucosidase, calmodulin, gars, glucose-6-phosphate dehydrogenase, heterogenous nuclear ribonucleoprotein A3, lon peptidase, nicotineamide adenine dinucleotide hydrogen (NADH) dehydrogenase, phosphoglycerate kinase, proteasome p45, rab2, pyruvate kinase and t-complex protein. The observed glucose-induced differential protein expression pattern indicates enhanced glucose metabolism, defense against reactive oxygen species, enhanced protein translation, folding and degradation and decreased insulin granular formation and trafficking. Palmitate-induced changes could be related to altered exocytosis. Conclusion The identified altered proteins indicate mechanism important for altered β-cell function in T2DM. PMID:19607692

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Efficient and accurate Greedy Search Methods for mining functional modules in protein interaction networks.

PubMed

He, Jieyue; Li, Chaojun; Ye, Baoliu; Zhong, Wei

2012-06-25

Most computational algorithms mainly focus on detecting highly connected subgraphs in PPI networks as protein complexes but ignore their inherent organization. Furthermore, many of these algorithms are computationally expensive. However, recent analysis indicates that experimentally detected protein complexes generally contain Core/attachment structures. In this paper, a Greedy Search Method based on Core-Attachment structure (GSM-CA) is proposed. The GSM-CA method detects densely connected regions in large protein-protein interaction networks based on the edge weight and two criteria for determining core nodes and attachment nodes. The GSM-CA method improves the prediction accuracy compared to other similar module detection approaches, however it is computationally expensive. Many module detection approaches are based on the traditional hierarchical methods, which is also computationally inefficient because the hierarchical tree structure produced by these approaches cannot provide adequate information to identify whether a network belongs to a module structure or not. In order to speed up the computational process, the Greedy Search Method based on Fast Clustering (GSM-FC) is proposed in this work. The edge weight based GSM-FC method uses a greedy procedure to traverse all edges just once to separate the network into the suitable set of modules. The proposed methods are applied to the protein interaction network of S. cerevisiae. Experimental results indicate that many significant functional modules are detected, most of which match the known complexes. Results also demonstrate that the GSM-FC algorithm is faster and more accurate as compared to other competing algorithms. Based on the new edge weight definition, the proposed algorithm takes advantages of the greedy search procedure to separate the network into the suitable set of modules. Experimental analysis shows that the identified modules are statistically significant. The algorithm can reduce the computational time significantly while keeping high prediction accuracy.

A Two-Week Guided Inquiry Protein Separation and Detection Experiment for Undergraduate Biochemistry

ERIC Educational Resources Information Center

Carolan, James P.; Nolta, Kathleen V.

2016-01-01

A laboratory experiment for teaching protein separation and detection in an undergraduate biochemistry laboratory course is described. This experiment, performed in two, 4 h laboratory periods, incorporates guided inquiry principles to introduce students to the concepts behind and difficulties of protein purification. After using size-exclusion…

Fabrication of superhydrophobic nano-aluminum films on stainless steel meshes by electrophoretic deposition for oil-water separation

NASA Astrophysics Data System (ADS)

Xu, Zhe; Jiang, Deyi; Wei, Zhibo; Chen, Jie; Jing, Jianfeng

2018-01-01

Stainless steel meshes with superhydrophobic surfaces were successfully fabricated via a facile electrophoretic deposition process. The surface morphology and chemical compositions were characterized by a field emission scanning electron microscope (FE-SEM), energy-dispersive X-ray spectroscope (EDS), X-ray diffraction (XRD) and fourier-transform infrared spectrophotometer (FTIR). After stearic acid modification, the obtained nano-aluminum films on stainless steel meshes showed an excellent superhydrophobic properties with a water contact angle of 160° ± 1.2° and a water sliding angle of less than 5°. In addition, on the basis of the superhydrophobic meshes, a simple, continuous oil-water separation apparatus was designed, and the oil-water separation efficiency was up to 95.8% ± 0.9%. Meanwhile, after 20 oil-water separation cycles, the separation efficiency without significant reduction suggested the stable performance of superhydrophobic stainless steel meshes on the oil-water separation. Moreover, the flow rate of oil-water mixture and effective separation length were investigated to determine their effects on the oil-water separation efficiency, respectively. Our work provides a cost-efficient method to prepare stable superhydrophobic nano-Al films on stainless steel meshes, and it has promising practical applications on oil-water separation.

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation

PubMed Central

Brady, Jacob P.; Farber, Patrick J.; Sekhar, Ashok; Lin, Yi-Hsuan; Huang, Rui; Bah, Alaji; Chan, Hue Sun; Forman-Kay, Julie D.; Kay, Lewis E.

2017-01-01

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid “membraneless organelles” that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, Ddx4cond, diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4cond to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4cond have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in Ddx4cond are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in Ddx4cond are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes. PMID:28894006

Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes.

PubMed

Baker, Perrin; Hillis, Colleen; Carere, Jason; Seah, Stephen Y K

2012-03-06

Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection

PubMed Central

Dickerson, Jane A.; Ramsay, Lauren M.; Dada, Oluwatosin O.; Cermak, Nathan

2011-01-01

Capillary isoelectric focusing and capillary zone electrophoresis are coupled with laser-induced fluorescence detection to create an ultrasensitive two-dimensional separation method for proteins. In this method, two capillaries are joined through a buffer filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second dimension separation. A fraction was transferred to the second dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125. PMID:20603830

CSE-MECC two-dimensional capillary electrophoresis analysis of proteins in the mouse tumor cell (AtT-20) homogenate

PubMed Central

Chen, Xingguo; Fazal, Md. Abul; Dovichi, Norman J.

2007-01-01

Two-dimensional capillary electrophoresis was used for the separation of proteins and biogenic amines from the mouse AtT-20 cell line. The first-dimension capillary contained a TRIS-CHES-SDS-dextran buffer to perform capillary sieving electrophoresis, which is based on molecular weight of proteins. The second-dimension capillary contained a TRIS-CHES-SDS buffer for micel1ar electrokinetic capillary chromatography. After a 61 seconds preliminary separation, fractions from the first-dimension capillary were successively transferred to the second-dimension capillary, where they further separated by MECC. The two-dimensional separation required 60 minutes. PMID:17637850

Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

PubMed Central

Smith, Jarrett; Calidas, Deepika; Schmidt, Helen; Lu, Tu; Rasoloson, Dominique; Seydoux, Geraldine

2016-01-01

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time. DOI: http://dx.doi.org/10.7554/eLife.21337.001 PMID:27914198

Separation of cancer cells from white blood cells by pinched flow fractionation.

PubMed

Pødenphant, Marie; Ashley, Neil; Koprowska, Kamila; Mir, Kalim U; Zalkovskij, Maksim; Bilenberg, Brian; Bodmer, Walter; Kristensen, Anders; Marie, Rodolphe

2015-12-21

In this paper, the microfluidic size-separation technique pinched flow fractionation (PFF) is used to separate cancer cells from white blood cells (WBCs). The cells are separated at efficiencies above 90% for both cell types. Circulating tumor cells (CTCs) are found in the blood of cancer patients and can form new tumors. CTCs are rare cells in blood, but they are important for the understanding of metastasis. There is therefore a high interest in developing a method for the enrichment of CTCs from blood samples, which also enables further analysis of the separated cells. The separation is challenged by the size overlap between cancer cells and the 10(6) times more abundant WBCs. The size overlap prevents high efficiency separation, however we demonstrate that cell deformability can be exploited in PFF devices to gain higher efficiencies than expected from the size distribution of the cells.

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.

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.

MimoSA: a system for minimotif annotation

PubMed Central

2010-01-01

Background Minimotifs are short peptide sequences within one protein, which are recognized by other proteins or molecules. While there are now several minimotif databases, they are incomplete. There are reports of many minimotifs in the primary literature, which have yet to be annotated, while entirely novel minimotifs continue to be published on a weekly basis. Our recently proposed function and sequence syntax for minimotifs enables us to build a general tool that will facilitate structured annotation and management of minimotif data from the biomedical literature. Results We have built the MimoSA application for minimotif annotation. The application supports management of the Minimotif Miner database, literature tracking, and annotation of new minimotifs. MimoSA enables the visualization, organization, selection and editing functions of minimotifs and their attributes in the MnM database. For the literature components, Mimosa provides paper status tracking and scoring of papers for annotation through a freely available machine learning approach, which is based on word correlation. The paper scoring algorithm is also available as a separate program, TextMine. Form-driven annotation of minimotif attributes enables entry of new minimotifs into the MnM database. Several supporting features increase the efficiency of annotation. The layered architecture of MimoSA allows for extensibility by separating the functions of paper scoring, minimotif visualization, and database management. MimoSA is readily adaptable to other annotation efforts that manually curate literature into a MySQL database. Conclusions MimoSA is an extensible application that facilitates minimotif annotation and integrates with the Minimotif Miner database. We have built MimoSA as an application that integrates dynamic abstract scoring with a high performance relational model of minimotif syntax. MimoSA's TextMine, an efficient paper-scoring algorithm, can be used to dynamically rank papers with respect to context. PMID:20565705

Conventional-Flow Liquid Chromatography-Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses.

PubMed

Lenčo, Juraj; Vajrychová, Marie; Pimková, Kristýna; Prokšová, Magdaléna; Benková, Markéta; Klimentová, Jana; Tambor, Vojtěch; Soukup, Ondřej

2018-04-17

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-μL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 μg of HeLa cells tryptic digest separated during a 60 min gradient at 68 μL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

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

Judd, R.C.; Caldwell, H.D.

1985-01-01

The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. Inmore » addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.« less

Distinguishing between Protein Dynamics and Dye Photophysics in Single-Molecule FRET Experiments

PubMed Central

Chung, Hoi Sung; Louis, John M.; Eaton, William A.

2010-01-01

Abstract Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small α/β protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers—donor10/acceptor57 and donor57/acceptor10—which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol–coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects—a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. PMID:20159166

Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.

PubMed

Chung, Hoi Sung; Louis, John M; Eaton, William A

2010-02-17

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Membrane protein separation and analysis by supercritical fluid chromatography-mass spectrometry.

PubMed

Zhang, Xu; Scalf, Mark; Westphall, Michael S; Smith, Lloyd M

2008-04-01

Membrane proteins comprise 25-30% of the human genome and play critical roles in a wide variety of important biological processes. However, their hydrophobic nature has compromised efforts at structural characterization by both X-ray crystallography and mass spectrometry. The detergents that are generally used to solubilize membrane proteins interfere with the crystallization process essential to X-ray studies and cause severe ion suppression effects that hinder mass spectrometric analysis. In this report, the use of supercritical fluid chromatography-mass spectrometry for the separation and analysis of integral membrane proteins and hydrophobic peptides is investigated. It is shown that detergents are rapidly and effectively separated from the proteins and peptides, yielding them in a state suitable for direct mass spectrometric analysis.

Development of pseudo-linear gradient elution for high-throughput resin selectivity screening in RoboColumn® Format.

PubMed

Kiesewetter, André; Menstell, Peter; Peeck, Lars H; Stein, Andreas

2016-11-01

Rapid development of chromatographic processes relies on effective high-throughput screening (HTS) methods. This article describes the development of pseudo-linear gradient elution for resin selectivity screening using RoboColumns ® . It gives guidelines for the implementation of this HTS method on a Tecan Freedom EVO ® robotic platform, addressing fundamental aspects of scale down and liquid handling. The creation of a flexible script for buffer preparation and column operation plus efficient data processing provided the basis for this work. Based on the concept of discretization, linear gradient elution was transformed into multistep gradients. The impact of column size, flow rate, multistep gradient design, and fractionation scheme on separation efficiency was systematically investigated, using a ternary model protein mixture. We identified key parameters and defined optimal settings for effective column performance. For proof of concept, we examined the selectivity of several cation exchange resins using various buffer conditions. The final protocol enabled a clear differentiation of resin selectivity on miniature chromatography column (MCC) scale. Distinct differences in separation behavior of individual resins and the influence of buffer conditions could be demonstrated. Results obtained with the robotic platform were representative and consistent with data generated on a conventional chromatography system. A study on antibody monomer/high molecular weight separation comparing MCC and lab scale under higher loading conditions provided evidence of the applicability of the miniaturized approach to practically relevant feedstocks with challenging separation tasks as well as of the predictive quality for larger scale. A comparison of varying degrees of robotic method complexity with corresponding effort (analysis time and labware consumption) and output quality highlights tradeoffs to select a method appropriate for a given separation challenge or analytical constraints. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1503-1519, 2016. © 2016 American Institute of Chemical Engineers.

System and process for efficient separation of biocrudes and water in a hydrothermal liquefaction system

DOEpatents

Elliott, Douglas C.; Hart, Todd R.; Neuenschwander, Gary G.; Oyler, James R.; Rotness, Jr, Leslie J.; Schmidt, Andrew J.; Zacher, Alan H.

2016-08-02

A system and process are described for clean separation of biocrudes and water by-products from hydrothermal liquefaction (HTL) product mixtures of organic and biomass-containing feedstocks at elevated temperatures and pressures. Inorganic compound solids are removed prior to separation of biocrude and water by-product fractions to minimize formation of emulsions that impede separation. Separation may be performed at higher temperatures that reduce heat loss and need to cool product mixtures to ambient. The present invention thus achieves separation efficiencies not achieved in conventional HTL processing.

Towards bedside washing of stored red blood cells: a prototype of a simple apparatus based on microscale sedimentation in normal gravity.

PubMed

Khanal, G; Huynh, R A; Torabian, K; Xia, H; Vörös, E; Shevkoplyas, S S

2018-01-01

Infusion of by-products of red blood cell (RBC) storage-induced degradation as well as of the residual plasma proteins and the anticoagulant-preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity-driven system for bedside washing of stored RBCs. Stored RBCs were diluted to 10% haematocrit (Hct) with normal saline, matching the conventional washing procedure. The dilute RBC suspensions were passed through a column of coiled tubing to allow RBC sedimentation in normal gravity, thus separating them from the washing solution. Washed RBCs were collected using bifurcations located along the tubing. Washing efficiency was quantified by measuring Hct, morphology, deformability, free haemoglobin and total-free protein. The gravity-driven washing system operating at 0·5 ml/min produced washed RBCs with final Hct of 36·7 ± 3·4% (32·3-41·2%, n = 10) and waste Hct of 3·4 ± 0·7% (2·4-4·3%, n = 10), while removing 80% of free haemoglobin and 90% of total-free protein. Washing improved the ability of stored RBCs to perfuse an artificial microvascular network by 20%. The efficiency of washing performed using the gravity-driven system was not significantly different than that of conventional centrifugation. This proof-of-concept study demonstrates the feasibility of washing stored RBCs using a simple, disposable system with efficiency comparable to that of conventional centrifugation, and thus represents a significant first step towards enabling low-cost washing of stored blood at bedside. © 2017 International Society of Blood Transfusion.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Electrical separation of protein concentrate from juice of forages. Final report

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

Koegel, R.G.; Straub, R.J.; McFate, K.L.

1993-03-01

Previous research has shown that large quantities of high-quality, low-fiber protein concentrate can be separated from the juice of forage crops such as alfalfa. The value of adding such extracted protein to the diet of undernourished children in Mexico and other developing countries has been well demonstrated. In the past, protein separation has been achieved by either heat coagulation of the protein or by a pH adjustment of the juice. Both techniques have disadvantages including irreversible changes in the protein and high energy or material costs. This used electrostatic fields to manipulate the small charges found in protein molecules. Suchmore » an approach could result in an on-farm or portable protein separation system that does not require the transport of large quantities of forage. Researchers, using a dc power supply with appropriately placed electrodes to separate protein from juices, varied voltage levels to modify field strength and tried various shapes of electrodes and configurations of apparatus. The relative impact of centrifugation, use of various flocculents, and ultrafiltration in attempts to enhance dc voltage-supply test results were explored. One steady-flow system used a plastic vessel with stainless steel walls that served as electrodes. Another steady-flow ac voltage system used a trough through which juice was allowed to flow While two spinning-disk electrodes passed electricity directly through the juice. A four-step process was developed using an, ac power supply. The juice is first treated with an ac current, then held for approximately 60 minutes, after which it is centrifuged at 10,000 g. In the final phase the soluble protein is concentrated 5--10 fold by ultrafiltration using filters with a 10,000 molecular weight cutoff. This process shows potential for meeting project objectives.« less

Rapid Protein Separations in Microfluidic Devices

NASA Technical Reports Server (NTRS)

Fan, Z. H.; Das, Champak; Xia, Zheng; Stoyanov, Alexander V.; Fredrickson, Carl K.

2004-01-01

This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

A new submarine oil-water separation system

NASA Astrophysics Data System (ADS)

Cai, Wen-Bin; Liu, Bo-Hong

2017-12-01

In order to solve the oil field losses of environmental problems and economic benefit caused by the separation of lifting production liquid to offshore platforms in the current offshore oil production, from the most basic separation principle, a new oil-water separation system has been processed of adsorption and desorption on related materials, achieving high efficiency and separation of oil and water phases. And the submarine oil-water separation device has been designed. The main structure of the device consists of gas-solid phase separation device, period separating device and adsorption device that completed high efficiency separation of oil, gas and water under the adsorption and desorption principle, and the processing capacity of the device is calculated.

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

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

Huang, Xin; Tian, Changhai; Liu, Miao

2012-04-06

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using thismore » platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.« less

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

PubMed

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeted Identification of Metastasis-associated Cell-surface Sialoglycoproteins in Prostate Cancer*

PubMed Central

Yang, Lifang; Nyalwidhe, Julius O.; Guo, Siqi; Drake, Richard R.; Semmes, O. John

2011-01-01

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells. PMID:21447706

Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

PubMed

Yang, Lifang; Nyalwidhe, Julius O; Guo, Siqi; Drake, Richard R; Semmes, O John

2011-06-01

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

Modeling phase separation in mixtures of intrinsically-disordered proteins

NASA Astrophysics Data System (ADS)

Gu, Chad; Zilman, Anton

Phase separation in a pure or mixed solution of intrinsically-disordered proteins (IDPs) and its role in various biological processes has generated interest from the theoretical biophysics community. Phase separation of IDPs has been implicated in the formation of membrane-less organelles such as nucleoli, as well as in a mechanism of selectivity in transport through the nuclear pore complex. Based on a lattice model of polymers, we study the phase diagram of IDPs in a mixture and describe the selective exclusion of soluble proteins from the dense-phase IDP aggregates. The model captures the essential behaviour of phase separation by a minimal set of coarse-grained parameters, corresponding to the average monomer-monomer and monomer-protein attraction strength, as well as the protein-to-monomer size ratio. Contrary to the intuition that strong monomer-monomer interaction increases exclusion of soluble proteins from the dense IDP aggregates, our model predicts that the concentration of soluble proteins in the aggregate phase as a function of monomer-monomer attraction is non-monotonic. We corroborate the predictions of the lattice model using Langevin dynamics simulations of grafted polymers in planar and cylindrical geometries, mimicking various in-vivo and in-vitro conditions.

A Modified MuDPIT Separation Identified 4,488 Proteins in a System Wide Analysis of Quiescence in Yeast

PubMed Central

Webb, Kristofor J.; Xu, Tao; Park, Sung Kyu; Yates, John R.

2013-01-01

A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near complete yeast proteome from a whole cell tryptic digest. This modified on-line two dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4,269 protein identifications were made from 4,189 distinguishable protein families from yeast during log phase growth. The “Micro” MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days’ time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations ‘oxidation reduction’, ‘catabolic processing’ and ‘cellular response to oxidative stress’ was seen in the quiescent cellular fraction, consistent with their long lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of ‘Ribosome’ and ‘Proteasome’, further defining the complex nature of yeast populations present during stationary phase growth. In total 4,488 distinguishable protein families were identified in all cellular conditions tested. PMID:23540446

Comparison of analytical protein separation characteristics for three amine-based capillary-channeled polymer (C-CP) stationary phases.

PubMed

Jiang, Liuwei; Marcus, R Kenneth

2016-02-01

Capillary-channeled polymer (C-CP) fiber stationary phases are finding utility in the realms of protein analytics as well as downstream processing. We have recently described the modification of poly(ethylene terephthalate) (PET) C-CP fibers to affect amine-rich phases for the weak anion-exchange (WAX) separation of proteins. Polyethylenimine (PEI) is covalently coupled to the PET surface, with subsequent cross-linking imparted by treatment with 1,4-butanediol diglycidyl ether (BUDGE). These modifications yield vastly improved dynamic binding capacities over the unmodified fibers. We have also previously employed native (unmodified) nylon 6 C-CP fibers as weak anion/cation-exchange (mixed-mode) and hydrophobic interaction chromatography (HIC) phases for protein separations. Polyamide, nylon 6, consists of amide groups along the polymer backbone, with primary amines and carboxylic acid end groups. The analytical separation characteristics of these three amine-based C-CP fiber phases are compared here. Each of the C-CP fiber columns in this study was shown to be able to separate a bovine serum albumin/hemoglobin/lysozyme mixture at high mobile phase linear velocity (∼70 mm s(-1)) but with different elution characteristics. These differences reflect the types of protein-surface interactions that are occurring, based on the active group composition of the fiber surfaces. This study provides important fundamental understanding for the development of surface-modified C-CP fiber columns for protein separation.

Real-time modulated nanoparticle separation with an ultra-large dynamic range.

PubMed

Zeming, Kerwin Kwek; Thakor, Nitish V; Zhang, Yong; Chen, Chia-Hung

2016-01-07

Nanoparticles exhibit size-dependent properties which make size-selective purification of proteins, DNA or synthetic nanoparticles essential for bio-analytics, clinical medicine, nano-plasmonics and nano-material sciences. Current purification methods of centrifugation, column chromatography and continuous-flow techniques suffer from particle aggregation, multi-stage process, complex setups and necessary nanofabrication. These increase process costs and time, reduce efficiency and limit dynamic range. Here, we achieve an unprecedented real-time nanoparticle separation (51-1500 nm) using a large-pore (2 μm) deterministic lateral displacement (DLD) device. No external force fields or nanofabrication are required. Instead, we investigated innate long-range electrostatic influences on nanoparticles within a fluid medium at different NaCl ionic concentrations. In this study we account for the electrostatic forces beyond Debye length and showed that they cannot be assumed as negligible especially for precise nanoparticle separation methods such as DLD. Our findings have enabled us to develop a model to simultaneously quantify and modulate the electrostatic force interactions between nanoparticle and micropore. By simply controlling buffer solutions, we achieve dynamic nanoparticle size separation on a single device with a rapid response time (<20 s) and an enlarged dynamic range (>1200%), outperforming standard benchtop centrifuge systems. This novel method and model combines device simplicity, isolation precision and dynamic flexibility, opening opportunities for high-throughput applications in nano-separation for industrial and biological applications.

Separation of replication and transcription domains in nucleoli.

PubMed

Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

2014-12-01

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species.

PubMed

Zheng, Songyan; Qiu, Difei; Adams, Monica; Li, Jinjiang; Mantri, Rao V; Gandhi, Rajesh

2017-01-01

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B 22 *, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

Multifunctional Nutrient-Binding Proteins Adapt Human Symbiotic Bacteria for Glycan Competition in the Gut by Separately Promoting Enhanced Sensing and Catalysis

PubMed Central

Cameron, Elizabeth A.; Kwiatkowski, Kurt J.; Lee, Byung-Hoo; Hamaker, Bruce R.; Koropatkin, Nicole M.

2014-01-01

ABSTRACT To compete for the dynamic stream of nutrients flowing into their ecosystem, colonic bacteria must respond rapidly to new resources and then catabolize them efficiently once they are detected. The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model for nutrient acquisition by symbiotic gut bacteria, which harbor thousands of related Sus-like systems. Structural investigation of the four Sus outer membrane proteins (SusD, -E, -F, and -G) revealed that they contain a total of eight starch-binding sites that we demonstrated, using genetic and biochemical approaches, to play distinct roles in starch metabolism in vitro and in vivo in gnotobiotic mice. SusD, whose homologs are abundant in the human microbiome, is critical for the initial sensing of available starch, allowing sus transcriptional activation at much lower concentrations than without this function. In contrast, seven additional binding sites across SusE, -F, and -G are dispensable for sus activation. However, they optimize the rate of growth on starch in a manner dependent on the expression of the bacterial polysaccharide capsule, suggesting that they have evolved to offset the diffusion barrier created by this structure. These findings demonstrate how proteins with similar biochemical behavior can serve orthogonal functions during different stages of cellular adaptation to nutrients. Finally, we demonstrated in gnotobiotic mice fed a starch-rich diet that the Sus binding sites confer a competitive advantage to B. thetaiotaomicron in vivo in a manner that is dependent on other colonizing microbes. This study reveals how numerically dominant families of carbohydrate-binding proteins in the human microbiome fulfill separate and sometimes cooperative roles to optimize gut commensal bacteria for nutrient acquisition. PMID:25205092

Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c.

PubMed

Roberts, V A; Freeman, H C; Olson, A J; Tainer, J A; Getzoff, E D

1991-07-15

To understand the specificity and efficiency of protein-protein interactions promoting electron transfer, we evaluated the role of electrostatic forces in precollision orientation by the development of two new methods, computer graphics alignment of protein electrostatic fields and a systematic orientational search of intermolecular electrostatic energies for two proteins at present separation distances. We applied these methods to the plastocyanin/cytochrome c interaction, which is faster than random collision, but too slow for study by molecular dynamics techniques. Significant electrostatic potentials were concentrated on one-fourth (969 A2) of the plastocyanin surface, with the greatest negative potential centered on the Tyr-83 hydroxyl within the acidic patch, and on one-eighth (632 A2) of the cytochrome c surface, with the greatest positive potential centered near the exposed heme edge. Coherent electrostatic fields occurred only over these regions, suggesting that local, rather than global, charge complementarity controls productive recognition. The three energetically favored families of pre-collision orientations all directed the positive region surrounding the heme edge of cytochrome c toward the acidic patch of plastocyanin but differed in heme plane orientation. Analysis of electrostatic fields, electrostatic energies of precollision orientations with 12 and 6 A separation distances, and surface topographies suggested that the favored orientations should converge to productive complexes promoting a single electron-transfer pathway from the cytochrome c heme edge to Tyr-83 of plastocyanin. Direct interactions of the exposed Cu ligand in plastocyanin with the cytochrome c heme edge are not unfavorable sterically or electrostatically but should occur no faster than randomly, indicating that this is not the primary pathway for electron transfer.

Analysis of Glycoproteins in Human Serum by Means of Glycospecific Magnetic Bead Separation and LC-MALDI-TOF/TOF Analysis with Automated Glycopeptide Detection

PubMed Central

Sparbier, Katrin; Asperger, Arndt; Resemann, Anja; Kessler, Irina; Koch, Sonja; Wenzel, Thomas; Stein, Günter; Vorwerg, Lars; Suckau, Detlev; Kostrzewa, Markus

2007-01-01

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively. PMID:17916798

Micro-flow injection system for the urinary protein assay.

PubMed

Nishihama, Syouhei; Imabayashi, Hisano; Matoba, Tomoko; Toya, Chika; Watanabe, Kosuke; Yoshizuka, Kazuharu

2008-02-15

A urinary protein assay has been investigated, employing a micro-flow injection analysis (muFIA) combined with an adsorptive separation of protein from analyte. The adsorptive separation part of protein in the artificial urine with ceramic hydroxyapatite is integrated on the muFIA chip, since the interference of other components coexisting in urine occurs in the conventional FIA system. The typical FI peak can be obtained following the adsorption-elution process of the protein prior to the detection, and the protein concentration in artificial urine can be quantitatively determined.

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain*

PubMed Central

Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Peter M.

2010-01-01

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method. PMID:20445002

Metallothionein quantification in clams by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization.

PubMed

Alhama, José; Romero-Ruiz, Antonio; López-Barea, Juan

2006-02-24

In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.

Computational biology approach to uncover hepatitis C virus helicase operation.

PubMed

Flechsig, Holger

2014-04-07

Hepatitis C virus (HCV) helicase is a molecular motor that splits nucleic acid duplex structures during viral replication, therefore representing a promising target for antiviral treatment. Hence, a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity. Despite extensive investigations performed in the past, a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved. Here we review investigations that have been previously performed by us for HCV helicase. Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation. We also discuss observations from that study in the light of recent experimental studies that confirm our findings.

Cell separation using tilted-angle standing surface acoustic waves

PubMed Central

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-01-01

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles–cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological–biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150

Cell separation using tilted-angle standing surface acoustic waves.

PubMed

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-09-09

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.

Functionalized Nano-adsorbent for Affinity Separation of Proteins

NASA Astrophysics Data System (ADS)

Zou, Xueyan; Yang, Fengbo; Sun, Xin; Qin, Mingming; Zhao, Yanbao; Zhang, Zhijun

2018-05-01

Thiol-functionalized silica nanospheres (SiO2-SH NSs) with an average diameter of 460 nm were synthesized through a hydrothermal route. Subsequently, the prepared SiO2-SH NSs were modified by SnO2 quantum dots to afford SnO2/SiO2 composite NSs possessing obvious fluorescence, which could be used to trace the target protein. The SnO2/SiO2 NSs were further modified by reduced glutathione (GSH) to obtain SnO2/SiO2-GSH NSs, which can specifically separate glutathione S-transferase-tagged (GST-tagged) protein. Moreover, the peroxidase activity of glutathione peroxidase 3 (GPX3) separated from SnO2/SiO2-GSH NSs in vitro was evaluated. Results show that the prepared SnO2/SiO2-GSH NSs exhibit negligible nonspecific adsorption, high concentration of protein binding (7.4 mg/g), and good reused properties. In the meantime, the GST-tagged GPX3 separated by these NSs can retain its redox state and peroxidase activity. Therefore, the prepared SnO2/SiO2-GSH NSs might find promising application in the rapid separation and purification of GST-tagged proteins.

Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE.

PubMed

Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan

2017-08-01

Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gold nanocage-based lateral flow immunoassay for immunoglobulin G

PubMed Central

Yang, Yunhui; Ozsoz, Mehmet

2017-01-01

The authors describe a gold nanocage-based lateral flow strip biosensor (LFSB) for low-cost and sensitive detection of IgG. This protein was used as a model analyte to demonstrate the proof-of-concept. The method combines the unique optical properties of gold nanocages (GNCs) with highly efficient chromatographic separation. A sandwich-type of immunoreactions occurs on the GNC-based LFSB which has the attractive features of avoiding multiple incubation, separation, and washing steps. The captured GNCs on the purple test zone and control zone of the biosensor are producing characteristic purple bands, and this enables IgG even to be visually detected. Quantitatation was accomplished by reading the intensities of the bands with a portable strip reader. The LFSB fabrication and assay parameters were optimized. The biosensor displays a linear response in the 0.5 to 50 ng·mL−1 IgG concentration range, and it has a 15 min assay time. The detection limit is 0.1 ng·mL−1 of IgG, which is 2.5 times lower than that when using a gold nanoparticle-based LFSB. In our perception, this assay has a wide potential for the detection of other proteins and species for which respective antibodies are available. PMID:29187761

Experimental design of a twin-column countercurrent gradient purification process.

PubMed

Steinebach, Fabian; Ulmer, Nicole; Decker, Lara; Aumann, Lars; Morbidelli, Massimo

2017-04-07

As typical for separation processes, single unit batch chromatography exhibits a trade-off between purity and yield. The twin-column MCSGP (multi-column countercurrent solvent gradient purification) process allows alleviating such trade-offs, particularly in the case of difficult separations. In this work an efficient and reliable procedure for the design of the twin-column MCSGP process is developed. This is based on a single batch chromatogram, which is selected as the design chromatogram. The derived MCSGP operation is not intended to provide optimal performance, but it provides the target product in the selected fraction of the batch chromatogram, but with higher yield. The design procedure is illustrated for the isolation of the main charge isoform of a monoclonal antibody from Protein A eluate with ion-exchange chromatography. The main charge isoform was obtained at a purity and yield larger than 90%. At the same time process related impurities such as HCP and leached Protein A as well as aggregates were at least equally well removed. Additionally, the impact of several design parameters on the process performance in terms of purity, yield, productivity and buffer consumption is discussed. The obtained results can be used for further fine-tuning of the process parameters so as to improve its performance. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

PubMed

Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

2013-12-01

Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient ( i.e ., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs . 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly ( p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold ( p = 0.0064) more abundant in HCR than LCR soleus. This discovery was verified using selective reaction monitoring (SRM) of the y5 ion (551.21 m/z ) of the doubly-charged peptide SLGVGFATR (454.19 m/z ) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater ( p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater ( p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for earlier transcriptome profiling work and show LC-MS is a viable means of profiling the abundance of almost all major metabolic enzymes of skeletal muscle in a highly parallel manner. Moreover, our approach is relatively more time efficient than techniques relying on orthogonal separations, and we demonstrate LC-MS profiling of the HCR/LCR selection model was able to highlight biomarkers that also exhibit differences in trained and untrained human muscle.

Non-invasive sex assessment in bovine semen by Raman spectroscopy

NASA Astrophysics Data System (ADS)

De Luca, A. C.; Managó, S.; Ferrara, M. A.; Rendina, I.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Ferraro, P.; Coppola, G.

2014-05-01

X- and Y-chromosome-bearing sperm cell sorting is of great interest, especially for animal production management systems and genetic improvement programs. Here, we demonstrate an optical method based on Raman spectroscopy to separate X- and Y-chromosome-bearing sperm cells, overcoming many of the limitations associated with current sex-sorting protocols. A priori Raman imaging of bull spermatozoa was utilized to select the sampling points (head-neck region), which were then used to discriminate cells based on a spectral classification model. Main variations of Raman peaks associated with the DNA content were observed together with a variation due to the sex membrane proteins. Next, we used principal component analysis to determine the efficiency of our device as a cell sorting method. The results (>90% accuracy) demonstrated that Raman spectroscopy is a powerful candidate for the development of a highly efficient, non-invasive, and non-destructive tool for sperm sexing.

Generation of chemical movies: FT-IR spectroscopic imaging of segmented flows.

PubMed

Chan, K L Andrew; Niu, X; deMello, A J; Kazarian, S G

2011-05-01

We have previously demonstrated that FT-IR spectroscopic imaging can be used as a powerful, label-free detection method for studying laminar flows. However, to date, the speed of image acquisition has been too slow for the efficient detection of moving droplets within segmented flow systems. In this paper, we demonstrate the extraction of fast FT-IR images with acquisition times of 50 ms. This approach allows efficient interrogation of segmented flow systems where aqueous droplets move at a speed of 2.5 mm/s. Consecutive FT-IR images separated by 120 ms intervals allow the generation of chemical movies at eight frames per second. The technique has been applied to the study of microfluidic systems containing moving droplets of water in oil and droplets of protein solution in oil. The presented work demonstrates the feasibility of the use of FT-IR imaging to study dynamic systems with subsecond temporal resolution.

Improved design of a tangential entry cyclone separator for separation of particles from exhaust gas of diesel engine.

PubMed

Mukhopadhyay, N

2011-01-01

An effective design of cyclone separator with tangential inlet is developed applying an equation derived from the correlation of collection efficiency with maximum pressure drop components of the cyclone, which can efficiently remove the particles around 1microm of the exhaust gas of diesel engine.

Oil-water separation property of polymer-contained wastewater from polymer-flooding oilfields in Bohai Bay, China.

PubMed

Chen, Hua-xing; Tang, Hong-ming; Duan, Ming; Liu, Yi-gang; Liu, Min; Zhao, Feng

2015-01-01

In this study, the effects of gravitational settling time, temperature, speed and time of centrifugation, flocculant type and dosage, bubble size and gas amount were investigated. The results show that the simple increase in settling time and temperature is of no use for oil-water separation of the three wastewater samples. As far as oil-water separation efficiency is concerned, increasing centrifugal speed and centrifugal time is highly effective for L sample, and has a certain effect on J sample, but is not valid for S sample. The flocculants are highly effective for S and L samples, and the oil-water separation efficiency increases with an increase in the concentration of inorganic cationic flocculants. There exist critical reagent concentrations for the organic cationic and the nonionic flocculants, wherein a higher or lower concentration of flocculant would cause a decrease in the treatment efficiency. Flotation is an effective approach for oil-water separation of polymer-contained wastewater from the three oilfields. The oil-water separation efficiency can be enhanced by increasing floatation agent concentration, flotation time and gas amount, and by decreasing bubble size.

Carbide derived carbon from MAX-phases and their separation applications

NASA Astrophysics Data System (ADS)

Hoffman, Elizabeth N.

Improved sorbents with increased selectivity and permeability are needed to meet growing energy and environmental needs. New forms of carbon based sorbents have been discovered recently, including carbons produced by etching metals from metal carbides, known as carbide derived carbons (CDCs). A common method for the synthesis of CDC is by chlorination at elevated temperatures. The goal of this work is to synthesize CDC from ternary carbides and to explore the links between the initial carbide chemistry and structure with the resulting CDCs properties, including porosity. CDC was produced from MAX-phase carbides, in particular Ti3SiC 2, Ti3AlC2, Ti2AlC, and Ta2AlC. Additionally, CDC was produced from Ta-based binary carbides, TaC and Ta 2C, and one carbo-nitride Ti2AlC0.5N0.5. The CDC structure was characterized using XRD, Raman microspectroscopy, and HRTEM. Porosity characterization was performed using sorption analysis with both Ar and N2 as adsorbates. It was determined the microporosity of CDC is related to the density of the initial carbide. The layered structure of the MAX-phase carbides lent toward the formation of larger mesopores within the resulting CDCs, while the amount of mesopores was dependent on the chemistry of the carbide. Furthermore, CDC produced from carbides with extremely high theoretical porosity resulted in small specific surface areas due to a collapse of the carbon structure. To expand the potential applications for CDC beyond powder and bulk forms, CDC membranes were produced from a thin film of TiC deposited by magnetron sputtering onto porous ceramic substrates. The TiC thin film was subsequently chlorinated to produce a bilayer membrane with CDC as the active layer. Both gases and liquids are capable of passing the membrane. The membrane separates based on selective adsorption, rather than a size separation molecular sieving effect. Two applications for CDC produced from MAX-phases were investigated: protein adsorption and gas separation. Sorbents capable of adsorbing large protein molecules efficiently are desirable for many medical applications, including the treatment of sepsis. Primarily mesoporous Ti2AlC-CDC and Ti3AlC2-CDC were proven to adsorb a significant amount of proteins compared to two current carbon adsorbents. When tested for gas separation, CDC was capable of selectively adsorbing gases including SF6, CO2, CH4, and H2. However, the gases were not separated based on their size, but rather on their interaction with the CDC surface.

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array.

PubMed

Shadpour, Hamed; Hupert, Mateusz L; Patterson, Donald; Liu, Changgeng; Galloway, Michelle; Stryjewski, Wieslaw; Goettert, Jost; Soper, Steven A

2007-02-01

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.

Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

PubMed

Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

2014-12-01

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

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. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks.

PubMed

Laube, Hendrik; Matysik, Frank-Michael; Schmidberger, Andreas; Mehlmann, Kerstin; Toursel, Andreas; Boden, Jana

2016-01-01

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system. A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount. The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-01

All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins.

The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides

PubMed Central

Tsirigos, Konstantinos D.; Peters, Christoph; Shu, Nanjiang; Käll, Lukas; Elofsson, Arne

2015-01-01

TOPCONS (http://topcons.net/) is a widely used web server for consensus prediction of membrane protein topology. We hereby present a major update to the server, with some substantial improvements, including the following: (i) TOPCONS can now efficiently separate signal peptides from transmembrane regions. (ii) The server can now differentiate more successfully between globular and membrane proteins. (iii) The server now is even slightly faster, although a much larger database is used to generate the multiple sequence alignments. For most proteins, the final prediction is produced in a matter of seconds. (iv) The user-friendly interface is retained, with the additional feature of submitting batch files and accessing the server programmatically using standard interfaces, making it thus ideal for proteome-wide analyses. Indicatively, the user can now scan the entire human proteome in a few days. (v) For proteins with homology to a known 3D structure, the homology-inferred topology is also displayed. (vi) Finally, the combination of methods currently implemented achieves an overall increase in performance by 4% as compared to the currently available best-scoring methods and TOPCONS is the only method that can identify signal peptides and still maintain a state-of-the-art performance in topology predictions. PMID:25969446

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

PubMed

Jiang, Xi-Wen; Wang, Jing; Chan, Leo Lai; Lam, Paul Kwan Sing; Gu, Ji-Dong

2015-08-01

Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

Enhancing Analytical Separations Using Super-Resolution Microscopy

NASA Astrophysics Data System (ADS)

Moringo, Nicholas A.; Shen, Hao; Bishop, Logan D. C.; Wang, Wenxiao; Landes, Christy F.

2018-04-01

Super-resolution microscopy is becoming an invaluable tool to investigate structure and dynamics driving protein interactions at interfaces. In this review, we highlight the applications of super-resolution microscopy for quantifying the physics and chemistry that occur between target proteins and stationary-phase supports during chromatographic separations. Our discussion concentrates on the newfound ability of super-resolved single-protein spectroscopy to inform theoretical parameters via quantification of adsorption-desorption dynamics, protein unfolding, and nanoconfined transport.

Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

2013-12-19

To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Integrated protein analysis platform based on column switch recycling size exclusion chromatography, microenzymatic reactor and microRPLC-ESI-MS/MS.

PubMed

Yuan, Huiming; Zhou, Yuan; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

2009-10-30

An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by microRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

PubMed

Ishihara, Takashi; Kadoya, Toshihiko; Endo, Naomi; Yamamoto, Shuichi

2006-05-05

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

The Unique Protein-to-Protein Carotenoid Transfer Mechanism.

PubMed

Maksimov, Eugene G; Sluchanko, Nikolai N; Slonimskiy, Yury B; Mironov, Kirill S; Klementiev, Konstantin E; Moldenhauer, Marcus; Friedrich, Thomas; Los, Dmitry A; Paschenko, Vladimir Z; Rubin, Andrew B

2017-07-25

Orange Carotenoid Protein (OCP) is known as an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for blue-green light absorbing keto-carotenoids, which excitation causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. Although recent studies greatly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced, to our knowledge, a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of photoactive OCP from completely photoinactive species. We accurately analyze the peculiarities of this process that, to the best of our knowledge, appears unique, a previously uncharacterized protein-to-protein carotenoid transfer mechanism. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

PubMed

Lukhovitskaya, Nina I; Cowan, Graham H; Vetukuri, Ramesh R; Tilsner, Jens; Torrance, Lesley; Savenkov, Eugene I

2015-03-01

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement. © 2015 American Society of Plant Biologists. All Rights Reserved.

Trophic transfer of trace metals: Subcellular compartmentalization in a polychaete and assimilation by a decapod crustacean

USGS Publications Warehouse

Rainbow, P.S.; Poirier, L.; Smith, B.D.; Brix, K.V.; Luoma, S.N.

2006-01-01

The chemical form of accumulated trace metal in prey is important in controlling the bioavailataility of dietary metal to a predator. This study investigated the trophic transfer of radiolabelled Ag, Cd and Zn from the polychaete worm Nereis diversicolor to the decapod crustacean Palaemonetes varians. We used 2 populations of worms with different proportions of accumulated metals in different subcellular fractions as prey, and loaded the worms with radiolabelled metals either from sediment or from solution. Accumulated radiolabelled metals were fractionated into 5 components : metal-rich granules (MRG), cellular debris, organelles, metallothionein-like proteins (MTLP), and other (heat-sensitive) proteins (HSP). Assimilation efficiencies (AE) of the metals by P. varians were measured from the 4 categories of prey (i.e. 2 populations, radiolabelled from sediment or solution). There were significant differences for each metal between the AEs from the different prey categories, confirming that origin of prey and route of uptake of accumulated trace metal will cause intraspecific differences in subsequent metal assimilation. Correlations were sought between AEs and selected fractions or combinations of fractions of metals in the prey-MRG, Trophically Available Metal (TAM = MTLP + HSP + organelles) and total protein (MTLP + HSP). TAM explained 28% of the variance in AEs for Ag, but no consistent relationships emerged between AEs and TAM or total protein when the metals were considered separately. AEs did, however, show significant positive regressions with both TAM and total protein when the 3 metals were considered together, explaining only about 21 % of the variance in each case. A significant negative relationship was observed between MRG and AE for all metals combined. The predator (P. varians) can assimilate dietary metal from a range of the fractions binding metals in the prey (N. diversicolor), with different assimilation efficiencies summated across these fractions. TAM and/or total protein may represent an approximate minimum for trophic availability but neither of these alone is a fully accurate predictor. ?? Inter-Research 2006.

Robust and durable superhydrophobic cotton fabrics for oil/water separation.

PubMed

Zhou, Xiaoyan; Zhang, Zhaozhu; Xu, Xianghui; Guo, Fang; Zhu, Xiaotao; Men, Xuehu; Ge, Bo

2013-08-14

By introducing the incorporation of polyaniline and fluorinated alkyl silane to the cotton fabric via a facile vapor phase deposition process, the fabric surface possessed superhydrophobicity with the water contact angle of 156° and superoleophilicity with the oil contact angle of 0°. The as-prepared fabric can be applied as effective materials for the separation of water and oil mixture with separation efficiency as high as 97.8%. Compared with other materials for oil/water separation, the reported process was simple, time-saving, and repeatable for at least 30 times. Moreover, the obtained fabric kept stable superhydrophobicity and high separation efficiency under extreme environment conditions of high temperature, high humidity, strong acidic or alkaline solutions, and mechanical forces. Therefore, this reported fabric has the advantages of scalable fabrication, high separation efficiency, stable recyclability, and excellent durability, exhibiting the strong potential for industrial production.

Vertical phase separation in bulk heterojunction solar cells formed by in situ polymerization of fulleride

PubMed Central

Zhang, Lipei; Xing, Xing; Zheng, Lingling; Chen, Zhijian; Xiao, Lixin; Qu, Bo; Gong, Qihuang

2014-01-01

Vertical phase separation of the donor and the acceptor in organic bulk heterojunction solar cells is crucial to improve the exciton dissociation and charge transport efficiencies. This is because whilst the exciton diffusion length is limited, the organic film must be thick enough to absorb sufficient light. However, it is still a challenge to control the phase separation of a binary blend in a bulk heterojunction device architecture. Here we report the realization of vertical phase separation induced by in situ photo-polymerization of the acrylate-based fulleride. The power conversion efficiency of the devices with vertical phase separation increased by 20%. By optimising the device architecture, the power conversion efficiency of the single junction device reached 8.47%. We believe that in situ photo-polymerization of acrylate-based fulleride is a universal and controllable way to realise vertical phase separation in organic blends. PMID:24861168

The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

PubMed

Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

2014-01-01

Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu. © 2014 American Institute of Chemical Engineers.

Facile Fabrication of a Polyethylene Mesh for Oil/Water Separation in a Complex Environment.

PubMed

Zhao, Tianyi; Zhang, Dongmei; Yu, Cunming; Jiang, Lei

2016-09-14

Low cost, eco-friendly, and easily scaled-up processes are needed to fabricate efficient oil/water separation materials, especially those useful in harsh environments such as highly acidic, alkaline, and salty environments, to deal with serious oil spills and industrial organic pollutants. Herein, a highly efficient oil/water separation mesh with durable chemical stability was fabricated by simply scratching and pricking a conventional polyethylene (PE) film. Multiscaled morphologies were obtained by this scratching and pricking process and provided the mesh with a special wettability performance termed superhydrophobicity, superoleophilicity, and low water adhesion, while the inert chemical properties of PE delivered chemical etching resistance to the fabricated mesh. In addition to a highly efficient oil/corrosive liquid separation, the fabricated PE mesh was also reusable and exhibited ultrafast oil/water separation solely by gravity. The easy operation, chemical durability, reusability, and efficiency of the novel PE mesh give it high potential for use in industrial and consumer applications.

Centrifugal precipitation chromatography

PubMed Central

Ito, Yoichiro; Lin, Qi

2009-01-01

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. The countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation. PMID:19541553

Rare earth separations by selective borate crystallization

PubMed Central

Yin, Xuemiao; Wang, Yaxing; Bai, Xiaojing; Wang, Yumin; Chen, Lanhua; Xiao, Chengliang; Diwu, Juan; Du, Shiyu; Chai, Zhifang; Albrecht-Schmitt, Thomas E.; Wang, Shuao

2017-01-01

Lanthanides possess similar chemical properties rendering their separation from one another a challenge of fundamental chemical and global importance given their incorporation into many advanced technologies. New separation strategies combining green chemistry with low cost and high efficiency remain highly desirable. We demonstrate that the subtle bonding differences among trivalent lanthanides can be amplified during the crystallization of borates, providing chemical recognition of specific lanthanides that originates from Ln3+ coordination alterations, borate polymerization diversity and soft ligand coordination selectivity. Six distinct phases are obtained under identical reaction conditions across lanthanide series, further leading to an efficient and cost-effective separation strategy via selective crystallization. As proof of concept, Nd/Sm and Nd/Dy are used as binary models to demonstrate solid/aqueous and solid/solid separation processes. Controlling the reaction kinetics gives rise to enhanced separation efficiency of Nd/Sm system and a one-step quantitative separation of Nd/Dy with the aid of selective density-based flotation. PMID:28290448

A robust TALENs system for highly efficient mammalian genome editing.

PubMed

Feng, Yuanxi; Zhang, Siliang; Huang, Xin

2014-01-10

Recently, transcription activator-like effector nucleases (TALENs) have emerged as a highly effective tool for genomic editing. A pair of TALENs binds to two DNA recognition sites separated by a spacer sequence, and the dimerized FokI nucleases at the C terminal then cleave DNA in the spacer. Because of its modular design and capacity to precisely target almost any desired genomic locus, TALEN is a technology that can revolutionize the entire biomedical research field. Currently, for genomic editing in cultured cells, two plasmids encoding a pair of TALENs are co-transfected, followed by limited dilution to isolate cell colonies with the intended genomic manipulation. However, uncertain transfection efficiency becomes a bottleneck, especially in hard-to-transfect cells, reducing the overall efficiency of genome editing. We have developed a robust TALENs system in which each TALEN plasmid also encodes a fluorescence protein. Thus, cells transfected with both TALEN plasmids, a prerequisite for genomic editing, can be isolated by fluorescence-activated cell sorting. Our improved TALENs system can be applied to all cultured cells to achieve highly efficient genomic editing. Furthermore, an optimized procedure for genomic editing using TALENs is also presented. We expect our system to be widely adopted by the scientific community.

Phylogeny of Immune Recognition: Processing and Presentation of Structurally Defined Proteins in Channel Catfish Immune Responses

PubMed Central

Vallejo, Abbe N.; Miller, Norman W.

1991-01-01

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cell subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104. PMID:1668258

Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis.

PubMed

Zhang, Bochao; Zhang, Xuewu

2013-01-01

Spirulina platensis is a multicellular edible blue-green alga with abundant proteins (∼ 60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze-thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF-7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5-FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2-CS) in food and pharmaceutical applications. © 2013 American Institute of Chemical Engineers.

On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum.

PubMed

Emara, Samy; Kamal, Maha; Abdel Kawi, Mohamed

2012-02-01

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated µ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated µ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts.

PubMed

Wrobel, Katarzyna; Kannamkumarath, Sasi S; Wrobel, Kazimierz; Caruso, Joseph A

2003-01-01

In this work, the use of methanesulfonic acid for protein hydrolysis is proposed for evaluation of Se-methionine in yeast, Brazil nuts, and possibly other selenium-rich biological samples. The hydrolysis was carried out by heating the sample with 4 mol L(-1) acid at reflux for 8 h. Two chromatographic techniques (size-exclusion and ion-pairing) coupled with ICP-MS detection were used to compare the release of Se-methionine from proteins by enzymatic (proteinase K, protease XIV) and acid hydrolyses. A more efficient liberation of Se-methionine was observed by acid hydrolysis. For quantification, the sample extracts were introduced onto a C8 Alltima column, and the separation was achieved with a mobile phase containing 5 mmol L(-1) hexanesulfonic acid in citrate buffer (pH 4.5)/methanol (95:5). The results obtained by standard addition showed 816+/-17 micro g g(-1) and 36.2+/-1.5 micro g g(-1) of selenium in the form of Se-methionine in yeast and nuts, respectively (65% and 75% of total selenium).

Design and construction of 2A peptide-linked multicistronic vectors.

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. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide 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. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategy.

PubMed

Korecká, Lucie; Jankovicová, Barbora; Krenková, Jana; Hernychová, Lenka; Slováková, Marcela; Le-Nell, Anne; Chmelik, Josef; Foret, Frantisek; Viovy, Jean-Louis; Bilková, Zusana

2008-02-01

We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon.

PubMed

Suzuki, Ryosuke; Ishikawa, Tomohiro; Konishi, Eiji; Matsuda, Mami; Watashi, Koichi; Aizaki, Hideki; Takasaki, Tomohiko; Wakita, Takaji

2014-01-01

A method for rapid production of single-round infectious particles (SRIPs) of flavivirus would be useful for viral mutagenesis studies. Here, we established a DNA-based production system for SRIPs of flavivirus. We constructed a Japanese encephalitis virus (JEV) subgenomic replicon plasmid, which lacked the C-prM-E (capsid-pre-membrane-envelope) coding region, under the control of the cytomegalovirus promoter. When the JEV replicon plasmid was transiently co-transfected with a JEV C-prM-E expression plasmid into 293T cells, SRIPs were produced, indicating successful trans-complementation with JEV structural proteins. Equivalent production levels were observed when C and prM-E proteins were provided separately. Furthermore, dengue types 1-4, West Nile, yellow fever or tick-borne encephalitis virus prM-E proteins could be utilized for production of chimaeric flavivirus SRIPs, although the production was less efficient for dengue and yellow fever viruses. These results indicated that our plasmid-based system is suitable for investigating the life cycles of flaviviruses, diagnostic applications and development of safer vaccine candidates.

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

PubMed Central

Piechura, Joseph R.; Tseng, Tzu-Ling; Hsu, Hsin-Fang; Byrne, Rose T.; Windgassen, Tricia A.; Chitteni-Pattu, Sindhu; Battista, John R.; Li, Hung-Wen; Cox, Michael M.

2015-01-01

Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism. PMID:25559557

Chromosome Missegregation Associated with RUVBL1 Deficiency

PubMed Central

Lauterbach, David; Gysi, Mario; Steigemann, Patrick; Gerlich, Daniel W.; Jiricny, Josef; Ferrari, Stefano

2015-01-01

RUVBL1 (RuvB-like1) and RUVBL2 (RuvB-like 2) are integral components of multisubunit protein complexes involved in processes ranging from cellular metabolism, transcription and chromatin remodeling to DNA repair. Here, we show that although RUVBL1 and RUVBL2 are known to form heterodimeric complexes in which they stabilize each other, the subunits separate during cytokinesis. In anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, where it partially co-localizes with polo-like kinase 1 (PLK1). The ability of PLK1 to phosphorylate RUVBL1—but not RUVBL2—in vitro and their physical association in vivo suggest that this kinase differentially regulates the function of the RuvB-like proteins during mitosis. We further show that siRNA-mediated knock-down of RuvB-like proteins causes severe defects in chromosome alignment and segregation. In addition, we show that the ATPase activity of RUVBL1 is indispensable for cell proliferation. Our data thus demonstrate that RUVBL1 is essential for efficient mitosis and proliferation. PMID:26201077

PIE-1 is a bifunctional protein that regulates maternal and zygotic gene expression in the embryonic germ line of Caenorhabditis elegans

PubMed Central

Tenenhaus, Christina; Subramaniam, Kuppuswamy; Dunn, Melanie A.; Seydoux, Geraldine

2001-01-01

The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage during the first cleavages of the Caenorhabditis elegans embryo. We have shown previously that one function of PIE-1 is to inhibit mRNA transcription. Here we show that PIE-1 has a second function in germ cells; it is required for efficient expression of the maternally encoded Nanos homolog NOS-2. This second function is genetically separable from PIE-1's inhibitory effect on transcription. A mutation in PIE-1's second CCCH finger reduces NOS-2 expression without affecting transcriptional repression and causes primordial germ cells to stray away from the somatic gonad, occasionally exiting the embryo entirely. Our results indicate that PIE-1 promotes germ cell fate by two independent mechanisms as follows: (1) inhibition of transcription, which blocks zygotic programs that drive somatic development, and (2) activation of protein expression from nos-2 and possibly other maternal RNAs, which promotes primordial germ cell development. PMID:11316796

Recent progress in preparation and application of microfluidic chip electrophoresis

NASA Astrophysics Data System (ADS)

Cong, Hailin; Xu, Xiaodan; Yu, Bing; Yuan, Hua; Peng, Qiaohong; Tian, Chao

2015-05-01

Since its discovery in 1990, microfluidic chip electrophoresis (MCE) has allowed the development of applications with small size, fast analysis, low cost, high integration density and automatic level, which are easy to carry and have made commercialization efficient. MCE has been widely used in the areas of environmental protection, biochemistry, medicine and health, clinical testing, judicial expertise, food sanitation, pharmaceutical checking, drug testing, agrochemistry, biomedical engineering and life science. As one of the foremost fields in the research of capillary electrophoresis, MCE is the ultimate frontier to develop the miniaturized, integrated, automated all-in-one instruments needed in modern analytical chemistry. By adopting the advanced technologies of micro-machining, lasers and microelectronics, and the latest research achievements in analytical chemistry and biochemistry, the sampling, separation and detection systems of commonly used capillary electrophoresis are integrated with high densities onto glass, quartz, silicon or polymer wafers to form the MCE, which can finish the analysis of multi-step operations such as injection, enrichment, reaction, derivatization, separation, and collection of samples in a portable, efficient and super high speed manner. With reference to the different technological achievements in this area, the latest developments in MCE are reviewed in this article. The preparation mechanisms, surface modifications, and properties of different materials in MCE are compared, and the different sampling, separation and detection systems in MCE are summarized. The performance of MCE in analysis of fluorescent substance, metallic ion, sugar, medicine, nucleic acid, DNA, amino acid, polypeptide and protein is discussed, and the future direction of development is forecast.

Accelerated molecular dynamics and protein conformational change: a theoretical and practical guide using a membrane embedded model neurotransmitter transporter.

PubMed

Gedeon, Patrick C; Thomas, James R; Madura, Jeffry D

2015-01-01

Molecular dynamics simulation provides a powerful and accurate method to model protein conformational change, yet timescale limitations often prevent direct assessment of the kinetic properties of interest. A large number of molecular dynamic steps are necessary for rare events to occur, which allow a system to overcome energy barriers and conformationally transition from one potential energy minimum to another. For many proteins, the energy landscape is further complicated by a multitude of potential energy wells, each separated by high free-energy barriers and each potentially representative of a functionally important protein conformation. To overcome these obstacles, accelerated molecular dynamics utilizes a robust bias potential function to simulate the transition between different potential energy minima. This straightforward approach more efficiently samples conformational space in comparison to classical molecular dynamics simulation, does not require advanced knowledge of the potential energy landscape and converges to the proper canonical distribution. Here, we review the theory behind accelerated molecular dynamics and discuss the approach in the context of modeling protein conformational change. As a practical example, we provide a detailed, step-by-step explanation of how to perform an accelerated molecular dynamics simulation using a model neurotransmitter transporter embedded in a lipid cell membrane. Changes in protein conformation of relevance to the substrate transport cycle are then examined using principle component analysis.

Reevaluating the mechanism of excitation energy regulation in iron-starved cyanobacteria.

PubMed

Chen, Hui-Yuan S; Liberton, Michelle; Pakrasi, Himadri B; Niedzwiedzki, Dariusz M

2017-03-01

This paper presents spectroscopic investigations of IsiA, a chlorophyll a-binding membrane protein produced by cyanobacteria grown in iron-deficient environments. IsiA, if associated with photosystem I, supports photosystem I in light harvesting by efficiently transferring excitation energy. However, if separated from photosystem I, IsiA exhibits considerable excitation quenching observed as a substantial reduction of protein-bound chlorophyll a fluorescence lifetime. Previous spectroscopic studies suggested that carotenoids are involved in excitation energy dissipation and in addition play a second role in this antenna complex by supporting chlorophyll a in light harvesting by absorbing in the spectral range inaccessible for chlorophyll a and transferring excitation to chlorophylls. However, this investigation does not support these proposed roles of carotenoids in this light harvesting protein. This study shows that carotenoids do not transfer excitation energy to chlorophyll a. In addition, our investigations do not support the hypothesis that carotenoids are quenchers of the excited state of chlorophyll a in this protein complex. We propose that quenching of chlorophyll a fluorescence in IsiA is maintained by pigment-protein interaction via electron transfer from an excited chlorophyll a to a cysteine residue, an excitation quenching mechanism that was recently proposed to regulate the light harvesting capabilities of the bacteriochlorophyll a-containing Fenna-Mathews-Olson protein from green sulfur bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

PubMed

Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

2013-11-01

Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Ultrahigh-Resolution Differential Ion Mobility Separations of Conformers for Proteins above 10 kDa: Onset of Dipole Alignment?

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

Shvartsburg, Alexandre A.

2014-11-04

Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with same peak width and resolving power up to ~400 as for peptides. Present work explores the reach of this approach for larger proteins, exemplified by cytochrome c and myoglobin. Resolution similar to thatmore » for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of present technique to proteins under ~20 kDa. This capability may enable distinguishing whole proteins with differing residue sequences or localizations of posttranslational modifications. Small features at negative compensation voltages that markedly grow from cytochrome c to myoglobin indicate the dipole alignment of rare conformers in accord with theory, further supporting the concept of pendular macroions in FAIMS.« less

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

PubMed

Naimuddin, Mohammed; Kubo, Tai

2011-12-01

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery. Copyright © 2011 Elsevier Inc. All rights reserved.

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.

PubMed

Kurena, Baiba; Vežāne, Aleksandra; Skrastiņa, Dace; Trofimova, Olga; Zajakina, Anna

2017-07-01

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media. Copyright © 2017 Elsevier B.V. All rights reserved.

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas.

PubMed

Franczyk, M; Lopucki, M; Stachowicz, N; Morawska, D; Kankofer, M

2017-02-01

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta. Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation. Copyright © 2016 Elsevier Ltd. All rights reserved.

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection.

PubMed

Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

2007-07-01

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.

Continuous, high-flux and efficient oil/water separation assisted by an integrated system with opposite wettability

NASA Astrophysics Data System (ADS)

Li, Jian; Long, Yifei; Xu, Changcheng; Tian, Haifeng; Wu, Yanxia; Zha, Fei

2018-03-01

To resolve the drawbacks that single-mesh involved for oil/water separation, such as batch processing mode, only one phase was purified and the quick decrease in flux et al., herein, a two-way separation T-tube device was designed by integrating a pair of meshes with opposite wettability, i.e., underwater superoleophobic and superhydrophobic/superoleophilic properties. Such integrated system can continuously separate both oil and water phase from the oil/water mixtures simultaneously through one-step procedure with high flux (above 3.675 L m-2 s-1) and high separation efficiency larger than 99.8% regardless of the heavy oil or light oil involved in the mixture. Moreover, the as-prepared two meshes still maintained high separation efficiency larger than above 98.9% even after 50 cycle-usages. It worthy mentioned that this two-way separation mode essentially solves the oil liquid accumulation problem that is the single separation membrane needs to tolerate a large hydrostatic pressure caused by the accumulated liquid. We deeply believe this two-way separation system would provide a new strategy for realizing practical applications in oil spill clean-up via a continuous mode.

Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

PubMed

Vincent, Delphine; Elkins, Aaron; Condina, Mark R; Ezernieks, Vilnis; Rochfort, Simone

2016-01-01

Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

Protein removal from waste brines generated during ham salting through acidification and centrifugation.

PubMed

Gutiérrez-Martínez, Maria del Rosario; Muñoz-Guerrero, Hernán; Alcaína-Miranda, Maria Isabel; Barat, José Manuel

2014-03-01

The salting step in food processes implies the production of large quantities of waste brines, having high organic load, high conductivity, and other pollutants with high oxygen demand. Direct disposal of the residual brine implies salinization of soil and eutrophication of water. Since most of the organic load of the waste brines comes from proteins leaked from the salted product, precipitation of dissolved proteins by acidification and removal by centrifugation is an operation to be used in waste brine cleaning. The aim of this study is optimizing the conditions for carrying out the separation of proteins from waste brines generated in the pork ham salting operation, by studying the influence of pH, centrifugal force, and centrifugation time. Models for determining the removal of proteins depending on the pH, centrifugal force, and time were obtained. The results showed a high efficacy of the proposed treatment for removing proteins, suggesting that this method could be used for waste brine protein removal. The best pH value to be used in an industrial process seems to be 3, while the obtained results indicate that almost 90% of the proteins from the brine can be removed by acidification followed by centrifugation. A further protein removal from the brine should have to be achieved using filtrating techniques, which efficiency could be highly improved as a consequence of the previous treatment through acidification and centrifugation. Waste brines from meat salting have high organic load and electrical conductivity. Proteins can be removed from the waste brine by acidification and centrifugation. The total protein removal can be up to 90% of the initial content of the waste brine. Protein removal is highly dependent on pH, centrifugation rate, and time. © 2014 Institute of Food Technologists®

Fabrication of superhydrophilic and underwater superoleophobic metal mesh by laser treatment and its application

NASA Astrophysics Data System (ADS)

Yu, Peng; Lian, Zhongxu; Xu, Jinkai; Yu, Zhanjiang; Ren, Wanfei; Yu, Huadong

2018-04-01

In this paper, a lot of micron-sized sand granular structures were formed on the substrate of the stainless steel mesh (SSM) by laser treatment. The rough surface with sand granular structures showed superhydrophilic in air and superoleophobic under water. With its special wettability, the SSM by laser treatment could achieve the separation of the oil/water mixture, showing good durability and high separation efficiency, which was very useful in the practical application of large-scale oil/water separation facility for reducing the impacts of oil leaked on the environment. In addition, it showed that the laser-treated SSM had a very high separation rate. The development of the laser-treated SSM is a simple, environmental, economical and high-efficiency method, which provides a new approach to the production of high efficiency facilities for oil/water separation.

[Polymorphism of genes encoding proteins of DNA repair vs. occupational and environmental exposure to lead, arsenic and pesticides].

PubMed

Bukowski, Karol; Woźniak, Katarzyna

2018-03-09

Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(2):225-235. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE-FPLC with ICP-(ORS)MS detection.

PubMed

Rodríguez-Cea, Andrés; de la Campa, María Rosario Fernández; Sanz-Medel, Alfredo

2005-01-01

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.

C-reactive protein and interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control.

PubMed

Zhao, Y; Czilwik, G; Klein, V; Mitsakakis, K; Zengerle, R; Paust, N

2017-05-02

We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on a disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept is composed of on demand ligand binding, two washing steps, the substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4% and residual liquid remaining within particles below 3%. The automated immunoassay concept was demonstrated by means of detecting C-reactive protein (CRP) in the range of 1-81 ng ml -1 and interleukin 6 (IL-6) in the range of 64-13 500 pg ml -1 . The limit of detection and quantification were 1.0 ng ml -1 and 2.1 ng ml -1 for CRP and 64 pg ml -1 and 205 pg ml -1 for IL-6, respectively.

Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

PubMed Central

Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

2014-01-01

Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

Rapidly separating microneedles for transdermal drug delivery.

PubMed

Zhu, Dan Dan; Wang, Qi Lei; Liu, Xu Bo; Guo, Xin Dong

2016-09-01

The applications of polymer microneedles (MNs) into human skin emerged as an alternative of the conventional hypodermic needles. However, dissolving MNs require many minutes to be dissolved in the skin and typically have difficulty being fully inserted into the skin, which may lead to the low drug delivery efficiency. To address these issues, we introduce rapidly separating MNs that can rapidly deliver drugs into the skin in a minimally invasive way. For the rapidly separating MNs, drug loaded dissolving MNs are mounted on the top of solid MNs, which are made of biodegradable polylactic acid which eliminate the biohazardous waste. These MNs have sufficient mechanical strength to be inserted into the skin with the drug loaded tips fully embedded for subsequent dissolution. Compared with the traditional MNs, rapidly separating MNs achieve over 90% of drug delivery efficiency in 30s while the traditional MNs needs 2min to achieve the same efficiency. With the in vivo test in mice, the micro-holes caused by rapidly separating MNs can heal in 1h, indicating that the rapidly separating MNs are safe for future applications. These results indicate that the design of rapidly separating dissolvable MNs can offer a quick, high efficient, convenient, safe and potentially self-administered method of drug delivery. Polymer microneedles offer an attractive, painless and minimally invasive approach for transdermal drug delivery. However, dissolving microneedles require many minutes to be dissolved in the skin and typically have difficulty being fully inserted into the skin due to the skin deformation, which may lead to the low drug delivery efficiency. In this work we proposed rapidly separating microneedles which can deliver over 90% of drug into the skin in 30s. The in vitro and in vivo results indicate that the new design of these microneedles can offer a quick, high efficient, convenient and safe method for transdermal drug delivery. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Structured crowding and its effects on enzyme catalysis.

PubMed

Ma, Buyong; Nussinov, Ruth

2013-01-01

Macromolecular crowding decreases the diffusion rate, shifts the equilibrium of protein-protein and protein-substrate interactions, and changes protein conformational dynamics. Collectively, these effects contribute to enzyme catalysis. Here we describe how crowding may bias the conformational change and dynamics of enzyme populations and in this way affect catalysis. Crowding effects have been studied using artificial crowding agents and in vivo-like environments. These studies revealed a correlation between protein dynamics and function in the crowded environment. We suggest that crowded environments be classified into uniform crowding and structured crowding. Uniform crowding represents random crowding conditions created by synthetic particles with a narrow size distribution. Structured crowding refers to the highly coordinated cellular environment, where proteins and other macromolecules are clustered and organized. In structured crowded environments the perturbation of protein thermal stability may be lower; however, it may still be able to modulate functions effectively and dynamically. Dynamic, allosteric enzymes could be more sensitive to cellular perturbations if their free energy landscape is flatter around the native state; on the other hand, if their free energy landscape is rougher, with high kinetic barriers separating deep minima, they could be more robust. Above all, cells are structured; and this holds both for the cytosol and for the membrane environment. The crowded environment is organized, which limits the search, and the crowders are not necessarily inert. More likely, they too transmit allosteric effects, and as such play important functional roles. Overall, structured cellular crowding may lead to higher enzyme efficiency and specificity.

Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins.

PubMed

Samalova, Marketa; Fricker, Mark; Moore, Ian

2006-12-01

Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the amino-terminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.

Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.

PubMed

Fang, Ying; Treffers, Emmely E; Li, Yanhua; Tas, Ali; Sun, Zhi; van der Meer, Yvonne; de Ru, Arnoud H; van Veelen, Peter A; Atkins, John F; Snijder, Eric J; Firth, Andrew E

2012-10-23

Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.

Effects of chemical composition of fly ash on efficiency of metal separation in ash-melting of municipal solid waste

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

Okada, Takashi, E-mail: t-okada@u-fukui.ac.jp; Tomikawa, Hiroki

2013-03-15

Highlights: ► Separation of Pb and Zn from Fe and Cu in ash-melting of municipal solid waste. ► Molar ratio of Cl to Na and K in fly ash affected the metal-separation efficiency. ► The low molar ratio and a non-oxidative atmosphere were better for the separation. - Abstract: In the process of metal separation by ash-melting, Fe and Cu in the incineration residue remain in the melting furnace as molten metal, whereas Pb and Zn in the residue are volatilized. This study investigated the effects of the chemical composition of incineration fly ash on the metal-separation efficiency of themore » ash-melting process. Incineration fly ash with different chemical compositions was melted with bottom ash in a lab-scale reactor, and the efficiency with which Pb and Zn were volatilized preventing the volatilization of Fe and Cu was evaluated. In addition, the behavior of these metals was simulated by thermodynamic equilibrium calculations. Depending on the exhaust gas treatment system used in the incinerator, the relationships among Na, K, and Cl concentrations in the incineration fly ash differed, which affected the efficiency of the metal separation. The amounts of Fe and Cu volatilized decreased by the decrease in the molar ratio of Cl to Na and K in the ash, promoting metal separation. The thermodynamic simulation predicted that the chlorination volatilization of Fe and Cu was prevented by the decrease in the molar ratio, as mentioned before. By melting incineration fly ash with the low molar ratio in a non-oxidative atmosphere, most of the Pb and Zn in the ash were volatilized leaving behind Fe and Cu.« less

Splitting the chromosome: cutting the ties that bind sister chromatids.

PubMed

Nasmyth, K; Peters, J M; Uhlmann, F

2001-01-01

In eukaryotic cells, replicated DNA molecules remain physically connected from their synthesis in S phase until they are separated during anaphase. This phenomenon, called sister chromatid cohesion, is essential for the temporal separation of DNA replication and mitosis and for the equal separation of the duplicated genome. Recent work has identified a number of chromosomal proteins required for cohesion. In this review we discuss how these proteins may connect sister chromatids and how they are removed from chromosomes to allow sister chromatid separation at the onset of anaphase.

Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

PubMed

Zhang, Liqun; Borthakur, Susmita; Buck, Matthias

2016-02-23

The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 μs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in that it initially involves transitions to surfaces with fewer, less favorable contacts compared with those seen in the fully formed complex. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Enrichment of peptides in serum by C(8)-functionalized magnetic nanoparticles for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

PubMed

Yao, Ning; Chen, Hemei; Lin, Huaqing; Deng, Chunhui; Zhang, Xiangmin

2008-03-21

Human serum contains a complex array of proteolytically derived peptides (serum peptidome), which contain biomarkers of preclinical screening and disease diagnosis. Recently, commercial C(8)-functionalized magnetic beads (1-10 microm) were widely applied to the separation and enrichment of peptides in human serum, prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. In this work, laboratory-prepared C(8)-functionalized magnetic nanoparticles (about 50 nm) were prepared and applied to the fast separation and the enrichment of peptides from serum. At first, the C(8)-magnetic nanoparticles were synthesized by modifying amine-functionalized magnetic nanoparticles with chlorodimethyloctylsilane. These synthesized C(8)-amine-functionalized magnetic particles have excellent magnetic responsibility, high dispersibility and large surface area. Finally, the C(8)-magnetic nanoparticles were successfully applied to fast and efficient enrichment of low-abundance peptides from protein tryptic digestion and human serum followed by MALDI-TOF-MS analysis.

Phase Separation in Solutions of Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

2012-02-01

We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

Fluidized-bed Fenton coupled with ceramic membrane separation for advanced treatment of flax wastewater.

PubMed

Fan, Dong; Ding, Lili; Huang, Hui; Chen, Mengtian; Ren, Hongqiang

2017-10-15

Fluidized-bed Fenton coupled with ceramic membrane separation to treat the flax secondary effluent was investigated. The operating variables, including initial pH, dosage of H 2 O 2 and Fe 0 , air flow rate, TMP and pore size, were optimized. The distributions of DOMs in the treatment process were analyzed. Under the optimum condition (600mgL -1 H 2 O 2 , 1.4gL -1 Fe 0 , pH=3, 300Lh -1 air flow rate and 15psi TMP), the highest TOC and color removal efficiencies were 84% and 94% in the coupled reactor with 100nm ceramic membrane, reducing 39% of total iron with similar removal efficiency compared with Fluidized-bed Fenton. Experimental results showed that the ceramic membrane could intercept catalyst particles (average particle size >100nm), 10.4% macromolecules organic matter (AMW>20000Da) and 12.53% hydrophobic humic-like component. EEM-PARAFAC identified four humic-like (M1-M4) and one protein-like components (M5), and the fluorescence intensities of M1-M5 in the secondary effluent were 63.27, 63.05, 33.41, 16.71 and 0.72 QSE, respectively. After the coupled treatment, the removal efficiencies of M1(81%), M2(86%) were higher than M3, M4(63%, 61%). Pearson correlation analysis suggested that M1, M2 and M3 were the major contributors to the cake layer, and M4, M5 might more easily lead to pore blockages. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplexed Western Blotting Using Microchip Electrophoresis.

PubMed

Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

2016-07-05

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomic biomarker discovery

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

Shi, Tujin; Zhou, Jianying; Gritsenko, Marina A.

2012-02-01

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up tomore » 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.« less

Attomole quantitation of protein separations with accelerator mass spectrometry

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

Vogel, J S; Grant, P G; Buccholz, B A

2000-12-15

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundancesmore » in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.« less

Infection of epithelial cells with dengue virus promotes the expression of proteins favoring the replication of certain viral strains.

PubMed

Martínez-Betancur, Viviana; Marín-Villa, Marcel; Martínez-Gutierrez, Marlén

2014-08-01

Dengue virus (DENV) is the causative agent of dengue and severe dengue. To understand better the dengue virus-host interaction, it is important to determine how the expression of cellular proteins is modified due to infection. Therefore, a comparison of protein expression was conducted in Vero cells infected with two different DENV strains, both serotype 2: DENV-2/NG (associated with dengue) and DENV-2/16681 (associated with severe dengue). The viability of the infected cells was determined, and neither strain induced cell death at 48 hr. In addition, the viral genomes and infectious viral particles were quantified, and the genome of the DENV-2/16681 strain was determined to have a higher replication rate compared with the DENV-2/NG strain. Finally, the proteins from infected and uninfected cultures were separated using two-dimensional gel electrophoresis, and the differentially expressed proteins were identified by mass spectrometry. Compared with the uninfected controls, the DENV-2/NG- and DENV-2/16681-infected cultures had five and six differentially expressed proteins, respectively. The most important results were observed when the infected cultures were compared to each other (DENV-2/NG vs. DENV-2/16681), and 18 differentially expressed proteins were identified. Based on their cellular functions, many of these proteins were linked to the increase in the replication efficiency of DENV. Among the proteins were calreticulin, acetyl coenzyme A, acetyl transferase, and fatty acid-binding protein. It was concluded that the infection of Vero cells with DENV-2/NG or DENV-2/16681 differentially modifies the expression of certain proteins, which can, in turn, facilitate infection. © 2013 Wiley Periodicals, Inc.

Microchip Electrophoresis at Elevated Temperatures and High Separation Field Strengths

PubMed Central

Mitra, Indranil; Marczak, Steven P.; Jacobson, Stephen C.

2014-01-01

We report free-solution microchip electrophoresis performed at elevated temperatures and high separation field strengths. We used microfluidic devices with 11-cm long separation channels to conduct separations at temperatures between 22 (ambient) and 45 °C and field strengths from 100 to 1000 V/cm. To evaluate separation performance, N-glycans were used as a model system and labeled with 8-aminopyrene-1,3,6-trisulfonic acid to impart charge for electrophoresis and render them fluorescent. Typically, increased diffusivity at higher temperatures leads to increased axial dispersion and poor separation performance; however, we demonstrate that sufficiently high separation field strengths can be used to offset the impact of increased diffusivity in order to maintain separation efficiency. Efficiencies for these free-solution separations are the same at temperatures of 25, 35, and 45 °C with separation field strengths ≥500 V/cm. PMID:24114979

Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

NASA Astrophysics Data System (ADS)

Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

2015-03-01

Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

The effect of protein on phase separation in giant unilamellar lipid vesicles.

NASA Astrophysics Data System (ADS)

Hutchison, J. B.; Weis, R. M.; Dinsmore, A. D.

2009-03-01

We explore the coarsening and out of plane curvature (budding) of domains in lipid bilayer vesicles composed of DOPC (unsaturated), PSM (saturated), and cholesterol. Green fluorescent protein (GFP) was added to the membrane in controlled amounts by binding to the Ni-chelating lipid, Ni-DOGS. Vesicles with diameters between 10 and 50 microns were prepared via a standard electroformation procedure. As a sample is lowered through temperature Tmix, a previously homogeneous vesicle phase separates into two fluid phases with distinct compositions. Phase-separated domains have a line tension (energy/length) at the boundary with the major phase which competes with bending energy and lateral tension to determine the overall configuration of the vesicle. Domain budding and coarsening were observed and recorded using both bright field and fluorescence microscopy during temperature scans and with varying concentrations of GFP. The addition of a model protein into our system allows for a broader understanding of the effect of protein, which are ubiquitous in cell membranes, on phase separation, budding, and coarsening.

Effect of protein properties on display efficiency using the M13 phage display system.

PubMed

Imai, S; Mukai, Y; Takeda, T; Abe, Y; Nagano, K; Kamada, H; Nakagawa, S; Tsunoda, S; Tsutsumi, Y

2008-10-01

The M13 phage display system is a powerful technology for engineering proteins such as functional mutant proteins and peptides. In this system, it is necessary that the protein is displayed on the phage surface. Therefore, its application is often limited when a protein is poorly displayed. In this study, we attempted to understand the relationship between a protein's properties and its display efficiency using the well-known pIII and pVIII type phage display system. The display of positively charged SV40 NLS and HIV-1 Tat peptides on pill was less efficient than that of the neutrally charged RGDS peptide. When different molecular weight proteins (1.5-58 kDa) were displayed on pIII and pVIII, their display efficiencies were directly influenced by their molecular weights. These results indicate the usefulness in predicting a desired protein's compatibility with protein and peptide engineering using the phage display system.

A novel, environmentally friendly sodium lauryl ether sulfate-, cocamidopropyl betaine-, cocamide monoethanolamine-containing buffer for MEKC on microfluidic devices.

PubMed

Hoeman, Kurt W; Culbertson, Christopher T

2008-12-01

A new buffer has been developed for fast, high-efficiency separations of amino acids by MEKC. This buffer was more environmentally friendly than the most commonly used surfactant-containing buffers for MEKC separations. It used a commercially available dishwashing soap by Seventh Generation (Burlington, VT, USA), which contained three micelle-forming agents. The mixed micelles were composed of sodium lauryl ether sulfate (anionic), cocamidopropyl betaine (zwitterionic), and cocamide monoethanolamine (non-ionic). The optimized buffer contained 5.0% w/w Seventh Generation Free & Clear dishwashing soap, 10 mM sodium borate, and was completely void of organics. The lack of organics and the biodegradability of the surfactant molecules made this buffer more environmentally friendly than typical SDS-containing buffers. This new buffer also had a different selectivity and provided faster separations with higher separation efficiencies than SDS-based buffers. Fast separations of BODIPY FL labeled amino acids yielded peaks with separation efficiencies greater than 100,000 in less than 20 s.

Separation of CHO cells using hydrocyclones.

PubMed

Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

2008-01-01

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Superhydrophobic meshes that can repel hot water and strong corrosive liquids used for efficient gravity-driven oil/water separation.

PubMed

Li, Jian; Kang, Ruimei; Tang, Xiaohua; She, Houde; Yang, Yaoxia; Zha, Fei

2016-04-14

Oil-polluted water has become a worldwide problem due to increasing industrial oily wastewater as well as frequent oil-spill pollution. Compared with underwater superoleophobic (water-removing) filtration membranes, superhydrophobic/superoleophilic (oil-removing) materials have advantages as they can be used for the filtration of heavy oil or the absorption of floating oil from water/oil mixtures. However, most of the superhydrophobic materials used for oil/water separation lose their superhydrophobicity when exposed to hot (e.g. >50 °C) water and strong corrosive liquids. Herein, we demonstrate superhydrophobic overlapped candle soot (CS) and silica coated meshes that can repel hot water (about 92 °C) and strong corrosive liquids, and were used for the gravity driven separation of oil-water mixtures in hot water and strong acidic, alkaline, and salty environments. To the best of our knowledge, we are unaware of any previously reported studies on the use of superhydrophobic materials for the separation of oil from hot water and corrosive aqueous media. In addition, the as-prepared robust superhydrophobic CS and silica coated meshes can separate a series of oils and organic solvents like kerosene, toluene, petroleum ether, heptane and chloroform from water with a separation efficiency larger than 99.0%. Moreover, the as-prepared coated mesh still maintained a separation efficiency above 98.5% and stable recyclability after 55 cycles of separation. The robust superhydrophobic meshes developed in this work can therefore be practically used as a highly efficient filtration membrane for the separation of oil from harsh water conditions, benefiting the environment and human health.

Highly efficient and ultra-small volume separation by pressure-driven liquid chromatography in extended nanochannels.

PubMed

Ishibashi, Ryo; Mawatari, Kazuma; Kitamori, Takehiko

2012-04-23

The rapidly developing interest in nanofluidic analysis, which is used to examine liquids ranging in amounts from the attoliter to the femtoliter scale, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. For general nanofluidic analysis, the fact that a pressure-driven flow does not limit the choice of solvents (aqueous or organic) is important. This study shows the first pressure-driven liquid chromatography technique that enables separation of atto- to femtoliter sample volumes, with a high separation efficiency within a few seconds. The apparent diffusion coefficient measurement of the unretentive sample suggests that there is no increase in the viscosity of toluene in the extended nanospace, unlike in aqueous solvents. Evaluation of the normal phase separation, therefore, should involve only the examination of the effect of the small size of the extended nanospace. Compared to a conventionally packed high-performance liquid chromatography column, the separation here results in a faster separation (4 s) by 2 orders of magnitude, a smaller injection volume (10(0) fL) by 9 orders, and a higher separation efficiency (440,000 plates/m) by 1 order. Moreover, the separation behavior agrees with the theory showing that this high efficiency was due to the small and controlled size of the separation channel, where the diffusion through the channel depth direction is fast enough to be neglected. Our chip-based platform should allow direct and real-time analysis or screening of ultralow volume of sample. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gas separation using ultrasound and light absorption

DOEpatents

Sinha, Dipen N [Los Alamos, NM

2012-07-31

An apparatus and method for separating a chosen gas from a mixture of gases having no moving parts and utilizing no chemical processing is described. The separation of particulates from fluid carriers thereof has been observed using ultrasound. In a similar manner, molecular species may be separated from carrier species. It is also known that light-induced drift may separate light-absorbing species from carrier species. Therefore, the combination of temporally pulsed absorption of light with ultrasonic concentration is expected to significantly increase the efficiency of separation by ultrasonic concentration alone. Additionally, breaking the spatial symmetry of a cylindrical acoustic concentrator decreases the spatial distribution of the concentrated particles, and increases the concentration efficiency.

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip.

PubMed

Tsai, Shuo-Wen; Loughran, Michael; Suzuki, Hiroaki; Karube, Isao

2004-02-01

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.