High-level Expression Purification Crystallization and Preliminary X-ray Crystallographic Studies of the Receptor Binding Domain of botulinum neurotoxin Serotype D

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

Y Zhang; X Gao; G Buchko

2011-12-31

Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less

High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

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

Zhang, Y.; Robinson, H.; Gao, X.

2010-12-01

Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

PubMed Central

Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

PubMed

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Online hyphenation of extraction, Sephadex LH-20 column chromatography, and high-speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step.

PubMed

Zhang, Ying-Qi; Wang, Shan-Shan; Han, Chao; Xu, Jin-Fang; Luo, Jian-Guang; Kong, Ling-Yi

2017-12-01

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH-20 column chromatography combined with high-speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH-20 column directly to cut the nontarget fractions followed by the second-dimensional high-speed countercurrent chromatography, hyphenated by a six-port valve equipped at the post-end of Sephadex LH-20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom.

PubMed

Garrett, Teresa A; Osmundson, Joseph; Isaacson, Marisa; Herrera, Jennifer

2015-01-01

In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project. © 2015 The International Union of Biochemistry and Molecular Biology.

Copper(I)/TEMPO Catalyzed Aerobic Oxidation of Primary Alcohols to Aldehydes with Ambient Air

PubMed Central

Hoover, Jessica M.; Steves, Janelle E.; Stahl, Shannon S.

2012-01-01

This protocol describes a practical laboratory-scale method for aerobic oxidation of primary alcohols to aldehydes, using a chemoselective CuI/TEMPO catalyst system. The catalyst is prepared in situ from commercially available reagents, and the reactions are performed in a common organic solvent (acetonitrile) with ambient air as the oxidant. Three different reaction conditions and three procedures for the isolation and purification of the aldehyde product are presented. The oxidations of eight different alcohols, described here, include representative examples of each reaction condition and purification method. Reaction times vary from 20 min to 24 h, depending on the alcohol, while the purification methods each take about 2 h. The total time necessary for the complete protocol ranges from 3 – 26 h. PMID:22635108

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

PubMed

Lee, Jin Suk

2017-01-01

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

General method for rapid purification of native chromatin fragments.

PubMed

Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A; Denu, John M

2018-05-24

Biochemical, proteomic and epigenetic studies of chromatin rely on the efficient ability to isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state, and is applicable for both parallel multi-sample spin-column purification and large scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes, features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy.  The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies, and as a mild, acid- and detergent-free sample preparation method for mass-spectrometry analysis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

PubMed

Franco-Medrano, Diana Ivonne; Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa María; Ortega-López, Jaime; Tejeda-Mansir, Armando

2017-03-01

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

PubMed

Buisson, C; Frelat, C; Mongongu, C; Martinat, N; Audran, M

2017-11-01

AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World Anti-Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation.

PubMed

Hansen, Marten; Varga, Eszter; Aarts, Cathelijn; Wust, Tatjana; Kuijpers, Taco; von Lindern, Marieke; van den Akker, Emile

2018-04-28

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Copyright © 2018. Published by Elsevier B.V.

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

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

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization.

PubMed

Vanz, Ana Ls; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-04-04

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

PubMed Central

Vanz, Ana LS; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-01-01

Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community. PMID:18394164

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

PubMed Central

Robert, Catherine; Pascalis, Hervé; Michelle, Caroline; Jardot, Priscilla; Charrel, Rémi; Raoult, Didier; Desnues, Christelle

2015-01-01

Background Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. PMID:26431175

Preparative two-step purification of recombinant H1.0 linker histone and its domains.

PubMed

Ivic, Nives; Bilokapic, Silvija; Halic, Mario

2017-01-01

H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay.

Rapid step-gradient purification of mitochondrial DNA.

PubMed

Welter, C; Meese, E; Blin, N

1988-01-01

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.

Diastereoselective chain-elongation reactions using microreactors for applications in complex molecule assembly.

PubMed

Carter, Catherine F; Lange, Heiko; Sakai, Daiki; Baxendale, Ian R; Ley, Steven V

2011-03-14

Diastereoselective chain-elongation reactions are important transformations for the assembly of complex molecular structures, such as those present in polyketide natural products. Here we report new methods for performing crotylation reactions and homopropargylation reactions by using newly developed low-temperature flow-chemistry technology. In-line purification protocols are described, as well as the application of the crotylation protocol in an automated multi-step sequence. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The purification step is not crucial in EIA measurements of thromboxane B2 and 11-dehydrothromboxane B2 in human plasma.

PubMed

Sadilkova, Lenka; Paluch, Zoltan; Mottlova, Jirina; Bednar, Frantisek; Alusik, Stefan

2012-01-01

Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes. For the purpose of this study, 20 plasma samples (10 healthy donors, 10 patients under treatment with acetylsalicylic acid) were screened for TxB2 and 11-dTxB2 concentrations using commercial competitive EIA kits (Cayman Chemicals, Tallinn, Estonia; Neogen, Lexington, KY, USA) with or without the introduction of the purification procedure. The purification step does not significantly affect the results of EIA measurements of the two of TxA2 metabolites (TxB2, 11-dTxB2) in human plasma. The levels of TxB2 and 11-dTxB2 determined in the plasma samples were not significantly changed (p < 0.05) when the purification step was omitted compared to the purified samples. This study establishes a protocol allowing for reliable and reproducible plasma TxB2 and 11-dTxB2 EIA measurement for routine basic screening of platelet function.

Analysis of translation using polysome profiling

PubMed Central

Chassé, Héloïse; Boulben, Sandrine; Costache, Vlad; Cormier, Patrick

2017-01-01

Abstract During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing. PMID:28180329

Antimicrobial Peptide Production and Purification.

PubMed

Suda, Srinivas; Field, Des; Barron, Niall

2017-01-01

Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

PubMed Central

Guyonnet, D.; Fremaux, C.; Cenatiempo, Y.; Berjeaud, J. M.

2000-01-01

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared. PMID:10742275

Purification of a Multidrug Resistance Transporter for Crystallization Studies

PubMed Central

Alegre, Kamela O.; Law, Christopher J.

2015-01-01

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

PubMed Central

Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

2016-01-01

Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator.

PubMed

Williams, Diana L; Adams, Linda B; Lahiri, Ramanuj

2014-10-01

Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue. Copyright © 2014. Published by Elsevier B.V.

Laboratory procedures to generate viral metagenomes.

PubMed

Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

2009-01-01

This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

DNA stable-isotope probing (DNA-SIP).

PubMed

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Metal- and additive-free photoinduced borylation of haloarenes.

PubMed

Mfuh, Adelphe M; Schneider, Brett D; Cruces, Westley; Larionov, Oleg V

2017-03-01

Boronic acids and esters have critical roles in the areas of synthetic organic chemistry, molecular sensors, materials science, drug discovery, and catalysis. Many of the current applications of boronic acids and esters require materials with very low levels of transition metal contamination. Most of the current methods for the synthesis of boronic acids, however, require transition metal catalysts and ligands that must be removed via additional purification procedures. This protocol describes a simple, metal- and additive-free method of conversion of haloarenes directly to boronic acids and esters. This photoinduced borylation protocol does not require expensive and toxic metal catalysts or ligands, and it produces innocuous and easy-to-remove by-products. Furthermore, the reaction can be carried out on multigram scales in common-grade solvents without the need for reaction mixtures to be deoxygenated. The setup and purification steps are typically accomplished within 1-3 h. The reactions can be run overnight, and the protocol can be completed within 13-16 h. Two representative procedures that are described in this protocol provide details for preparation of a boronic acid (3-cyanopheylboronic acid) and a boronic ester (1,4-benzenediboronic acid bis(pinacol)ester). We also discuss additional details of the method that will be helpful in the application of the protocol to other haloarene substrates.

Purification of boron nitride nanotubes via polymer wrapping

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

Choi, Jin-Hyuk; Kim, Jaewoo; WCI Quantum Beam based Radiation Research Center, Korea Atomic Energy Research Institute, 1045 Daedukdaero, Daejeon 305-353

2013-03-15

Highlights: ► Surface modification of boron nitride nanotubes using polymeric materials. ► Surface-modified BNNT was purified with a simple dilution-centrifugation step. ► Surface-modified BNNT can be directly used for polymer composite fabrication ► Degree of purification was analyzed by Raman spectroscopy. - Abstract: Boron nitride nanotubes (BNNT) synthesized by a ball milling-annealing were surface-modified using three different types of polymeric materials. Those materials were chosen depending on future applications especially in polymer nanocomposite fabrications. We found that the surface-modified BNNT can be purified with a simple dilution-centrifugation step, which would be suitable for large-scale purification. Degree of purification was monitoredmore » by means of the center peak position and FWHM of E{sub 2g} mode of BNNT in Raman spectra. As the purification of BNNT develops, the peak position was up-shifted while FWHM of the peak was narrowed.« less

Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

PubMed

Solomon, R G; Appels, R

1999-06-01

The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Synthesis and purification of 1,3,5-triamino-2,4,6-trinitrobenzene (TATB)

DOEpatents

Mitchell, Alexander R [Livermore, CA; Coburn, Michael D [Santa Fe, NM; Lee, Gregory S [San Ramon, CA; Schmidt, Robert D [Livermore, CA; Pagoria, Philip F [Livermore, CA; Hsu, Peter C [Pleasanton, CA

2006-06-06

A method to convert surplus nitroarene explosives (picric acid, ammonium picrate,) into TATB is described. The process comprises three major steps: conversion of picric acid/ammonium picrate into picramide; conversion of picramide to TATB through vicarious nucleophilic substitution (VNS) of hydrogen chemistry; and purification of TATB.

Experimental Optimal Single Qubit Purification in an NMR Quantum Information Processor

PubMed Central

Hou, Shi-Yao; Sheng, Yu-Bo; Feng, Guan-Ru; Long, Gui-Lu

2014-01-01

High quality single qubits are the building blocks in quantum information processing. But they are vulnerable to environmental noise. To overcome noise, purification techniques, which generate qubits with higher purities from qubits with lower purities, have been proposed. Purifications have attracted much interest and been widely studied. However, the full experimental demonstration of an optimal single qubit purification protocol proposed by Cirac, Ekert and Macchiavello [Phys. Rev. Lett. 82, 4344 (1999), the CEM protocol] more than one and half decades ago, still remains an experimental challenge, as it requires more complicated networks and a higher level of precision controls. In this work, we design an experiment scheme that realizes the CEM protocol with explicit symmetrization of the wave functions. The purification scheme was successfully implemented in a nuclear magnetic resonance quantum information processor. The experiment fully demonstrated the purification protocol, and showed that it is an effective way of protecting qubits against errors and decoherence. PMID:25358758

Development of a purification procedure for the isolation of nucleosides from urine prior to mass spectrometric analysis.

PubMed

Dudley, E; El-Shakawi, S; Games, D E; Newton, R P

2000-03-01

A chromatographic separation of nucleosides from urine has been developed in order to facilitate their mass spectrometric analysis for clinical diagnosis. A number of chromatographic resins were studied in order to develop an effective and efficient purification procedure. The optimized sequential protocol comprises a centrifugation, acidification and neutralization step, followed by application of an affinity chromatographic column and finally further separation on an acidic cation exchange column and a basic anion exchanger. This scheme shows effective clean-up of a standard radiolabelled nucleoside with a recovery of 92.5%, and recovery of nucleosides added to urine samples before extraction showed recoveries of 72-82%.

Purification and Characterization of the Bacterial Flagellar Basal Body from Salmonella enterica.

PubMed

Aizawa, Shin-Ichi

2017-01-01

The bacterial flagellum is a motility organelle. The flagellum is composed of three main structures: the basal body as a rotary engine embedded in the cellular membranes and cell wall, the long external filament that acts as a propeller, and the hook acting as a universal joint that connects them. I describe protocols for the purification of the filament and hook-basal body from Salmonella enterica serovar Typhimurium.

Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies.

PubMed

Dąbrowska, Krystyna

2018-01-01

In all cases when a bacteriophage makes direct contact with a mammalian organism, it may challenge the mammalian immunological system. Its major consequence is production of antibodies specific to the bacteriophage. Here we present protocols applicable in studies of bacteriophage ability to induce specific antibodies. The protocols have been divided into three parts: purification, immunization, and detection (ELISA).

Partitioning of human and sheep forms of the pathogenic prion protein during the purification of therapeutic proteins from human plasma.

PubMed

Stenland, Christopher J; Lee, Douglas C; Brown, Paul; Petteway, Stephen R; Rubenstein, Richard

2002-11-01

Therapeutic proteins derived from human plasma and other biologic sources have demonstrated an excellent safety record relative to the potential threat of transmissible spongiform encephalopathy (TSE) transmission. Previously, hamster-adapted scrapie was used as a model agent to assess TSE clearance in purification steps leading to the isolation of biopharmaceutical proteins. The current study investigated the validity of hamster scrapie as a model for human TSE clearance studies. The partitioning of the pathogenic forms of the prion protein associated with human variant CJD (PrP(vCJD)), human sporadic CJD (PrP(sCJD)) and Gerstmann-Sträussler-Scheinker (PrP(GSS)) syndrome was compared to the partitioning of hamster scrapie (PrP(Sc)) in three plasma protein purification steps. Sheep scrapie (PrP(Sc)) was similarly evaluated. The starting materials for three plasma protein purification steps, cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation, were spiked with brain homogenates containing human PrP(vCJD), human PrP(sCJD), human PrP(GSS), sheep PrP(Sc), and hamster 263K PrP(Sc). The partitioning of the pathogenic form of the PrP was analyzed. Clearance of the pathogenic form of the PrP was measured relative to the effluent fraction. Regardless of the source of the pathogenic prion, clearance was similar to hamster PrP(Sc). A nominal amount of clearance (approx., 1 log), an intermediate amount of clearance (approx., 2 log), and a substantial amount of clearance (> or = 3 log) were observed for the cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation steps, respectively. In the latter step, no PrP was detected in the effluents. These data demonstrate that human prions, including vCJD prions, can be removed during the purification of human therapeutic proteins and indicate that partitioning of human prions is similar to that observed in the hamster scrapie model.

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

Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch; Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève; Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yieldedmore » 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic –activated cell sorting step.« less

Three-Step Synthesis of Chiral Spirocyclic Oxaphospholenes.

PubMed

Berton, Jan K E T; Salemi, Hadi; Pirat, Jean-Luc; Virieux, David; Stevens, Christian V

2017-12-01

Chiral spirocylic oxaphospholenes were prepared in a three-step sequence from chiral pool terpenoid ketones. After addition of a metal acetylide, the resulting propargyl alcohols were converted stereoselectively into their allenylphosphonate counterparts. In the last step, they were conveniently cyclized into spirooxaphospholenes with one equivalent of iodine without purification. When starting from sterically hindered terpenes, allenylphosphonates were also easily obtained but showed to be unreactive or rearranged under these cyclization conditions.

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

PubMed

Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

2017-01-01

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

Strep-Tagged Protein Purification.

PubMed

Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

2015-01-01

The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

One-step purification of assembly-competent tubulin from diverse eukaryotic sources

PubMed Central

Widlund, Per O.; Podolski, Marija; Reber, Simone; Alper, Joshua; Storch, Marko; Hyman, Anthony A.; Howard, Jonathon; Drechsel, David N.

2012-01-01

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. PMID:22993214

Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation.

PubMed

Shore, Sabrina; Henderson, Jordana M; Lebedev, Alexandre; Salcedo, Michelle P; Zon, Gerald; McCaffrey, Anton P; Paul, Natasha; Hogrefe, Richard I

2016-01-01

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.

Removal and recovery of acetic acid and two furans during sugar purification of simulated phenols-free biomass hydrolysates.

PubMed

Lee, Sang Cheol

2017-12-01

A cost-effective five-step sugar purification process involving simultaneous removal and recovery of fermentation inhibitors from biomass hydrolysates was first proposed here. Only the three separation steps (PB, PC and PD) in the process were investigated here. Furfural was selectively removed up to 98.4% from a simulated five-component hydrolysate in a cross-current three-stage extraction system with n-hexane. Most of acetic acid in a simulated four-component hydrolysate was selectively removed by emulsion liquid membrane, and it could be concentrated in the stripping solution up to 4.5 times its initial concentration in the feed solution. 5-Hydroxymethylfurfural was selectively removed from a simulated three-component hydrolysate in batch and continuous fixed-bed column adsorption systems with L-493 adsorbent. Also, 5-hydroxymethylfurfural could be concentrated to about 9 times its feed concentration in the continuous adsorption system through a fixed-bed column desorption experiment with aqueous ethanol solution. These results have shown that the proposed purification process was valid. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experimental purification of single qubits.

PubMed

Ricci, M; De Martini, F; Cerf, N J; Filip, R; Fiurásek, J; Macchiavello, C

2004-10-22

We report the experimental realization of the purification protocol for single qubits sent through a depolarizing channel. The qubits are associated with polarization states of single photons and the protocol is achieved by means of passive linear optical elements. The present approach may represent a convenient alternative to the distillation and error correction protocols of quantum information.

Simple proof of security of the BB84 quantum key distribution protocol

PubMed

Shor; Preskill

2000-07-10

We prove that the 1984 protocol of Bennett and Brassard (BB84) for quantum key distribution is secure. We first give a key distribution protocol based on entanglement purification, which can be proven secure using methods from Lo and Chau's proof of security for a similar protocol. We then show that the security of this protocol implies the security of BB84. The entanglement purification based protocol uses Calderbank-Shor-Steane codes, and properties of these codes are used to remove the use of quantum computation from the Lo-Chau protocol.

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high-speed counter-current chromatography.

PubMed

Gao, Xiang; Zhuang, Rongqiang; Guo, Jiannan; Bao, Jian; Fang, Meijuan; Liu, Yan; Xu, Pengxiang; Zhao, Yufen

2010-02-01

In this paper, high-speed counter-current chromatography (HSCCC), assisted with ESI-MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7'-O-formylbrefeldin A (6.5 mg, 95.0%) and 7'-O-acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK-15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two-step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two-step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n-hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X-ray crystallography, ESI-MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.

An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

PubMed

Verma, Digvijay; Satyanarayana, T

2011-09-01

An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

Generation of insulin-producing cells from human bone marrow-derived mesenchymal stem cells: comparison of three differentiation protocols.

PubMed

Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; El-Badri, Nagwa; Ghoneim, Mohamed A

2014-01-01

Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs) to form insulin-producing cells (IPCs). We compared the relative efficiency of three differentiation protocols. Human bone marrow-derived MSCs (HBM-MSCs) were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol), trichostatin-A-based (two-step protocol), and β -mercaptoethanol-based (three-step protocol). At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. By immunolabeling, the proportion of generated IPCs was modest ( ≃ 3%) in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

Natriuretic Hormone: The Ultimate Determinant of the Preservation of External Sodium Balance

PubMed Central

Bricker, Neal S.; Cain, Christopher D.; Shankel, Stewart

2014-01-01

The present manuscript focuses on a putative natriuretic hormone. It includes the history of a long-term search for the pure molecule, ranging from partial purification to synthesis. It includes a description of seven different bioassay systems used, a resume of the sequential steps in purification, and a summary of a series of experimental protocols employed in the effort to define the biologic properties of the inhibitor of sodium (Na) transport. Two closely related molecules were purified and synthesized. Both are xanthurenic acid derivatives (xanthurenic acid 8-O-β-D-glucoside and xanthurenic acid 8-O-sulfate). It is concluded that one or both of these two low molecular weight compounds (MW: 368 and 284) meet many of the criteria for the final modulator of Na excretion. PMID:25566186

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

PubMed

Dhir, Sunny; Walia, Yashika; Zaidi, A A; Hallan, Vipin

2015-03-01

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

PubMed Central

Hennig, Bianca P.; Velten, Lars; Racke, Ines; Tu, Chelsea Szu; Thoms, Matthias; Rybin, Vladimir; Besir, Hüseyin; Remans, Kim; Steinmetz, Lars M.

2017-01-01

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing. PMID:29118030

RNA interactome capture in yeast.

PubMed

Beckmann, Benedikt M

2017-04-15

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol. Copyright © 2016. Published by Elsevier Inc.

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

PubMed Central

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

2011-01-01

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

Analysis of the proteolysis of bioactive peptides using a peptidomics approach

PubMed Central

Kim, Yun-Gon; Lone, Anna Mari; Saghatelian, Alan

2014-01-01

Identifying the peptidases that inactivate bioactive peptides (e.g. peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that combines liquid chromatography-mass spectrometry peptidomics to identify endogenous cleavage sites of a bioactive peptide, the subsequent biochemical purification of a candidate peptidase based on these cleavage sites, and validation of the candidate peptidase’s role in the physiological regulation of the bioactive peptide by examining a peptidase knockout mouse. We highlight successful application of this protocol to discover that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels and detail the key stages and steps in this approach. This protocol requires 7 days of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents, namely purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics. PMID:23949379

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

PubMed

Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

2017-03-17

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Bacterial and fungal DNA extraction from blood samples: automated protocols.

PubMed

Lorenz, Michael G; Disqué, Claudia; Mühl, Helge

2015-01-01

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

Analysis of In Vivo Chromatin and Protein Interactions of Arabidopsis Transcript Elongation Factors.

PubMed

Pfab, Alexander; Antosz, Wojciech; Holzinger, Philipp; Bruckmann, Astrid; Griesenbeck, Joachim; Grasser, Klaus D

2017-01-01

A central step to elucidate the function of proteins commonly comprises the analysis of their molecular interactions in vivo. For nuclear regulatory proteins this involves determining protein-protein interactions as well as mapping of chromatin binding sites. Here, we present two protocols to identify protein-protein and chromatin interactions of transcript elongation factors (TEFs) in Arabidopsis. The first protocol (Subheading 3.1) describes protein affinity-purification coupled to mass spectrometry (AP-MS) that utilizes suspension cultured cells as experimental system. This approach provides an unbiased view of proteins interacting with epitope-tagged TEFs. The second protocol (Subheading 3.2) depicts details about a chromatin immunoprecipitation (ChIP) procedure to characterize genomic binding sites of TEFs. These methods should be valuable tools for the analysis of a broad variety of nuclear proteins.

A gradient-free method for the purification of infective dengue virus for protein-level investigations.

PubMed

Jensen, Stephanie M; Nguyen, Celina T; Jewett, John C

2016-09-01

Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22.

PubMed

Li, Qian; Chang, Young-Tae

2006-01-01

This protocol outlines a methodology for the preparation and characterization of three RNA-specific fluorescent probes (E36, E144 and F22) and their use in live cell imaging. It describes a detailed procedure for their chemical synthesis and purification; serial product characterization and quality control tests, including measurements of their fluorescence properties in solution, measurement of RNA specificity and analysis of cellular toxicity; and live cell staining and counterstaining with Hoechst or DAPI. Preparation and application of these RNA imaging probes takes 1 week.

Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation.

PubMed

Hammerschmidt, Nikolaus; Tscheliessnig, Anne; Sommer, Ralf; Helk, Bernhard; Jungbauer, Alois

2014-06-01

Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

PubMed

Mackin, Robert B

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

PubMed Central

Mackin, Robert B.

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix. PMID:26150942

Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)

PubMed Central

El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaâ; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-01-01

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances. PMID:24124454

One-step separation and purification of three lignans and one flavonol from Sinopodophyllum emodi by medium-pressure liquid chromatography and high-speed counter-current chromatography.

PubMed

Wang, Ping; Liu, Yongling; Chen, Tao; Xu, Wenhua; You, Jinmao; Liu, Yongjun; Li, Yulin

2013-01-01

Lignans and flavonols are the primary constituents of Sinopodophyllum emodi and have been used as cathartic, anthelmintic, chemotherapeutic and anti-hypertensive compounds. Although these compounds have been isolated, there have been no reports on the separation of 4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin and kaempferol in one step by medium-pressure liquid chromatography (MPLC) and high-speed counter-current chromatography (HSCCC). Development of an efficient method for the preparative separation and purification of three lignans and one flavonol from S. emodi. The precipitate of crude extracts was first separated by MPLC into four parts, numbered GJ-1, GJ-2, GJ-3 and GJ-4. GJ-1 was separated and purified by HSCCC using a solvent system composed of n-hexane:ethyl acetate:methanol:water (1.75:1.5:1:0.75, v/v/v/v). The purities of the target compounds were assessed using high-performance liquid chromatography (HPLC) and chemical structures were identified by (1) H-NMR and (13) C-NMR. The HSCCC and MPLC methods were successfully used for the preparative separation and purification of 4'-demethyl podophyllotoxin (8.5 mg, 92.4%), podophyllotoxin (40.1 mg, 92.1%), deoxypodophyllotoxin (4.6 mg, 98.1%), and kaempferol (1.6 mg, 96.7%) from a 100 mg sample. Three lignans (4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin) and one flavonol (kaempferol) were successfully isolated by HSCCC and MPLC in one step. Copyright © 2013 John Wiley & Sons, Ltd.

Facile MALDI-MS analysis of neutral glycans in NaOH-doped matrixes: microwave-assisted deglycosylation and one-step purification with diamond nanoparticles.

PubMed

Tzeng, Yan-Kai; Chang, Cheng-Chun; Huang, Chien-Ning; Wu, Chih-Che; Han, Chau-Chung; Chang, Huan-Cheng

2008-09-01

A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5-dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B.

Automated high-throughput purification of genomic DNA from plant leaf or seed using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2004-06-01

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology"s Invader methods.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

NASA Astrophysics Data System (ADS)

Lane, Rebecca E.; Korbie, Darren; Anderson, Will; Vaidyanathan, Ramanathan; Trau, Matt

2015-01-01

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

Purification of crude glycerol from transesterification reaction of palm oil using direct method and multistep method

NASA Astrophysics Data System (ADS)

Nasir, N. F.; Mirus, M. F.; Ismail, M.

2017-09-01

Crude glycerol which produced from transesterification reaction has limited usage if it does not undergo purification process. It also contains excess methanol, catalyst and soap. Conventionally, purification method of the crude glycerol involves high cost and complex processes. This study aimed to determine the effects of using different purification methods which are direct method (comprises of ion exchange and methanol removal steps) and multistep method (comprises of neutralization, filtration, ion exchange and methanol removal steps). Two crude glycerol samples were investigated; the self-produced sample through the transesterification process of palm oil and the sample obtained from biodiesel plant. Samples were analysed using Fourier Transform Infrared Spectroscopy, Gas Chromatography and High Performance Liquid Chromatography. The results of this study for both samples after purification have showed that the pure glycerol was successfully produced and fatty acid salts were eliminated. Also, the results indicated the absence of methanol in both samples after purification process. In short, the combination of 4 purification steps has contributed to a higher quality of glycerol. Multistep purification method gave a better result compared to the direct method as neutralization and filtration steps helped in removing most excess salt, fatty acid and catalyst.

Facile Purification of Milligram to Gram Quantities of Condensed Tannins According to Mean Degree of Polymerization and Flavan-3-ol Subunit Composition.

PubMed

Brown, Ron H; Mueller-Harvey, Irene; Zeller, Wayne E; Reinhardt, Laurie; Stringano, Elisabetta; Gea, An; Drake, Christopher; Ropiak, Honorata M; Fryganas, Christos; Ramsay, Aina; Hardcastle, Emily E

2017-09-13

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1 H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1 H- 13 C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

Early process development of API applied to poorly water-soluble TBID.

PubMed

Meise, Marius; Niggemann, Matthias; Dunens, Alexandra; Schoenitz, Martin; Kuschnerow, Jan C; Kunick, Conrad; Scholl, Stephan

2018-05-01

Finding and optimising of synthesis processes for active pharmaceutical ingredients (API) is time consuming. In the finding phase, established methods for synthesis, purification and formulation are used to achieve a high purity API for biological studies. For promising API candidates, this is followed by pre-clinical and clinical studies requiring sufficient quantities of the active component. Ideally, these should be produced with a process representative for a later production process and suitable for scaling to production capacity. This work presents an overview of different approaches for process synthesis based on an existing lab protocol. This is demonstrated for the production of the model drug 4,5,6,7-tetrabromo-2-(1H-imidazol-2-yl) isoindolin-1,3-dione (TBID). Early batch synthesis and purification procedures typically suffer from low and fluctuating yields and purities due to poor process control. In a first step the literature synthesis and purification procedure was modified and optimized using solubility measurements, targeting easier and safer processing for consecutive studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Soft-Bake Purification of SWCNTs Produced by Pulsed Laser Vaporization

NASA Technical Reports Server (NTRS)

Yowell, Leonard; Nikolaev, Pavel; Gorelik, Olga; Allada, Rama Kumar; Sosa, Edward; Arepalli, Sivaram

2013-01-01

The "soft-bake" method is a simple and reliable initial purification step first proposed by researchers at Rice University for single-walled carbon nanotubes (SWCNT) produced by high-pressure carbon mon oxide disproportionation (HiPco). Soft-baking consists of annealing as-produced (raw) SWCNT, at low temperatures in humid air, in order to degrade the heavy graphitic shells that surround metal particle impurities. Once these shells are cracked open by the expansion and slow oxidation of the metal particles, the metal impurities can be digested through treatment with hydrochloric acid. The soft-baking of SWCNT produced by pulsed-laser vaporization (PLV) is not straightforward, because the larger average SWCNT diameters (.1.4 nm) and heavier graphitic shells surrounding metal particles call for increased temperatures during soft-bake. A part of the technology development focused on optimizing the temperature so that effective cracking of the graphitic shells is balanced with maintaining a reasonable yield, which was a critical aspect of this study. Once the ideal temperature was determined, a number of samples of raw SWCNT were purified using the soft-bake method. An important benefit to this process is the reduced time and effort required for soft-bake versus the standard purification route for SWCNT. The total time spent purifying samples by soft-bake is one week per batch, which equates to a factor of three reduction in the time required for purification as compared to the standard acid purification method. Reduction of the number of steps also appears to be an important factor in improving reproducibility of yield and purity of SWCNT, as small deviations are likely to get amplified over the course of a complicated multi-step purification process.

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.

PubMed

Albrecht, Melanie; Alessandri, Stefano; Conti, Amedeo; Reuter, Andreas; Lauer, Iris; Vieths, Stefan; Reese, Gerald

2008-11-01

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

PubMed

Dong, Dexian; Gui, Yanli; Chen, Dezhao; Li, Rongxiu

2008-01-01

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. 2008 John Wiley & Sons, Ltd

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation.

PubMed

Allen, Joselyn N; Dey, Adwitia; Nissly, Ruth; Fraser, James; Yu, Shan; Balandaram, Gayathri; Peters, Jeffrey M; Hankey-Giblin, Pamela A

2017-04-03

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds

PubMed Central

Sondermeijer, Hugo P; Witkowski, Piotr; Woodland, David; Seki, Tetsunori; Aangenendt, Frank J; van der Laarse, Arnoud; Itescu, Silviu; Hardy, Mark A

2017-01-01

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 μg/g, and RNA by 61% down to less than 10 μg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria. PMID:27114440

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

PubMed

Lee, Seon-Hwa; Hong, Seung-Hye; Kim, Kyoung-Rok; Oh, Deok-Kun

2017-08-01

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Fructose at 1 M (180 g l -1 ) was converted to 0.8 M (144 g l -1 ) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l -1 h -1 . No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Crystallization of Membrane Proteins by Vapor Diffusion

PubMed Central

Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

2016-01-01

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

PubMed

Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

2014-06-01

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Improving the large scale purification of the HIV microbicide, griffithsin.

PubMed

Fuqua, Joshua L; Wanga, Valentine; Palmer, Kenneth E

2015-02-22

Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.

A Two-Step Synthesis of Virstatin, a Virulence Inhibitor of "Vibrio cholerae"

ERIC Educational Resources Information Center

McDonald, Chriss E.

2009-01-01

Virstatin, an "N"-butanoic acid substituted naphthalimide, inhibits the ability of "Vibrio cholerae" to cause disease. A three-week experiment involving synthesis, purification, and spectral characterization of this compound is described. This experiment is appropriate for organic chemistry. It has been performed with three lab sections of about…

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

PubMed Central

Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

2015-01-01

Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

Purification of white spot syndrome virus by iodixanol density gradient centrifugation.

PubMed

Dantas-Lima, J J; Corteel, M; Cornelissen, M; Bossier, P; Sorgeloos, P; Nauwynck, H J

2013-10-01

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures. © 2013 John Wiley & Sons Ltd.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator.

PubMed

Costa-Borges, Nuno; Bellés, Marta; Meseguer, Marcos; Galliano, Daniela; Ballesteros, Agustin; Calderón, Gloria

2016-03-01

To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). Prospective cohort study on sibling donor oocytes. University-affiliated in vitro fertilization (IVF) center. Embryos from 59 patients. Culture in a TLI in a single medium with or without renewal of the medium on day-3. Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Treatment influencing down-staging in EORTC Melanoma Group sentinel node histological protocol compared with complete step-sectioning: a national multicentre study.

PubMed

Riber-Hansen, Rikke; Hastrup, Nina; Clemmensen, Ole; Behrendt, Nille; Klausen, Siri; Ramsing, Mette; Spaun, Eva; Hamilton-Dutoit, Stephen Jacques; Steiniche, Torben

2012-02-01

Metastasis size in melanoma sentinel lymph nodes (SLNs) is an emerging prognostic factor. Two European melanoma treatment trials include SLN metastasis diameters as inclusion criteria. Whilst diameter estimates are sensitive to the number of sections examined, the level of this bias is largely unknown. We performed a prospective multicentre study to compare the European Organisation for Research and Treatment of Cancer (EORTC) recommended protocol with a protocol of complete step-sectioning. One hundred and thirty-three consecutive SLNs from seven SLN centres were analysed by five central sections 50μm apart (EORTC Protocol) followed by complete 250μm step-sectioning. Overall, 29 patients (21.8%) were SLN-positive. The EORTC Protocol missed eight of these metastases (28%), one metastasis measuring less than 0.1mm in diameter, seven measuring between 0.1 and 1mm. Complete step-sectioning at 250μm intervals (Extensive Protocol) missed one metastasis (3%) that measured less than 0.1mm. Thirteen treatment courses (34%) performed if inclusion was based on the Combined Protocol would not be performed if assessed by the EORTC Protocol. Thus, 10 patients would be without completion lymph node dissection (EORTC MINITUB study), whilst three patients would not be eligible for anti-CTLA4 trial (EORTC protocol 18071). The corresponding number with the Extensive Protocol would be three; one patient for the MINITUB registration study and two patients for the anti-CTLA4 study. Examining SLNs by close central sectioning alone (EORTC Protocol) misses a substantial number of metastases and underestimates the maximum metastasis diameter, leading to important changes in patient eligibility for various treatment protocols. Copyright © 2011 Elsevier Ltd. All rights reserved.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences

PubMed Central

Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L.

2017-01-01

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. PMID:28628204

Secretory immunoglobulin purification from whey by chromatographic techniques.

PubMed

Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

2017-08-15

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification of functionalized DNA origami nanostructures.

PubMed

Shaw, Alan; Benson, Erik; Högberg, Björn

2015-05-26

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

Ultrasound-assisted three-phase partitioning of polyphenol oxidase from potato peel (Solanum tuberosum).

PubMed

Niphadkar, Sonali S; Rathod, Virendra K

2015-01-01

Conventional three phase partitioning (TPP) and ultrasound assisted three phase partitioning (UATPP) were optimized for achieving the maximum extraction and purification of polyphenol oxidase (PPO) from waste potato peels. Different process parameters such as ammonium sulfate (NH4)2SO4 concentration, crude extract to t-butanol ratio, time, temperature and pH were studied for conventional TPP. Except agitation speed, the similar parameters were also optimized for UATPP. Further additional parameters were also studied for UATPP viz. irradiation time at different frequencies, duty cycle and, rated power in order to obtain the maximum purification factor and recovery of PPO. The optimized conditions for conventional TPP were (NH4)2SO4 0-40% (w/v), extract to t-butanol ratio 1:1 (v/v), time 40 min and pH 7 at 30°C. These conditions provided 6.3 purification factor and 70% recovery of PPO from bottom phase. On the other hand, UATPP gives maximum purification fold of 19.7 with 98.3% recovery under optimized parameters which includes (NH4)2SO4 0-40% (w/v), crude extract to t-butanol ratio 1: 1 (v/v) pH 7, irradiation time 5 min with 25 kHz, duty cycle 40% and rated power 150W at 30°C. UATPP delivers higher purification factor and % recovery of PPO along with reduced operation time from 40 min to 5 min when compared with TPP. SDS PAGE showed partial purification of PPO enzyme with UATPP with molecular weight in the range of 26-36 kDa. Results reveal that UATPP would be an attractive option for the isolation and purification of PPO without need of multiple steps. © 2015 American Institute of Chemical Engineers.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification of Logic-Qubit Entanglement.

PubMed

Zhou, Lan; Sheng, Yu-Bo

2016-07-05

Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network.

Oxime-based linker libraries as a general approach for the rapid generation and screening of multidentate inhibitors

PubMed Central

Bahta, Medhanit; Liu, Fa; Kim, Sung-Eun; Stephen, Andrew G.; Fisher, Robert J.; Burke, Terrence R.

2013-01-01

The described oxime-based library protocol provides detailed procedures for the linkage of aminooxy functionality with aldehyde building blocks that result in the generation of libraries of multidentate inhibitors. Synthesis of inhibitors for protein tyrosine phosphatases (PTPs) and antagonists directed against the human tumor susceptibility gene 101 (Tsg101) are shown as examples. Three steps are involved: a) the design and synthesis of aminooxy platforms; b) tethering with aldehydes to form oxime-based linkages with sufficient purity; and c) direct in vitro biological evaluation of oxime products without purification. Each coupling reaction is a) performed in capped microtubes at room temperature; b) diluted for inhibitory evaluation and c) screened with targets in microplates to provide IC50 or Kd values. The synthesis of the aminooxy platforms takes 3–5 days; tethering with the aldehydes takes 24 h; and inhibition assay of enzymes and protein-protein interactions (PPIs) takes 30 min and 2 h respectively. PMID:22422315

Biomarkers of organophosphorus (OP) exposures in humans

PubMed Central

Marsillach, Judit; Richter, Rebecca J.; Kim, Jerry H.; Stevens, Richard C.; MacCoss, Michael J.; Tomazela, Daniela; Suzuki, Stephanie M.; Schopfer, Lawrence M; Lockridge, Oksana; Furlong, Clement E.

2011-01-01

There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of “biomarker proteins” that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS. PMID:21767566

Biomarkers of organophosphorus (OP) exposures in humans.

PubMed

Marsillach, Judit; Richter, Rebecca J; Kim, Jerry H; Stevens, Richard C; MacCoss, Michael J; Tomazela, Daniela; Suzuki, Stephanie M; Schopfer, Lawrence M; Lockridge, Oksana; Furlong, Clement E

2011-10-01

There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS. Copyright © 2011 Elsevier Inc. All rights reserved.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

PubMed

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

PubMed

Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

2015-05-01

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography.

PubMed

Gao, Min; Gu, Ming; Liu, Chun-Zhao

2006-07-11

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

The development of a purification procedure for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D; Fenske, B; Owen, T G; Shyng, S

1995-02-01

A simple economical procedure for purifying saxitoxin-induced protein (SIP) from crude extracts of the small shore crab, Hemigrapsus oregenesis, was developed. (NH4)2SO4 precipitation, chymotrypsin digestion, heat treatment, gel filtration and ion-exchange-chromatography procedures were evaluated in purifying SIP. An enzyme immunoassay was used to determine the SIP yield and relative purity at each step of three procedures, thus permitting an assessment of the conditions required for maximum recovery. Response surface analysis was used in an attempt to determine the optimum temperature and exposure time for the heat treatment. A 20 min incubation at 65 degrees C was confirmed by electrophoretic analysis to be the best combination of time and temperature for achieving both an acceptable yield and purity of SIP. SIP in desalted concentrate was shown to be resistant to chymotrypsin proteolysis; however, this enzyme had deleterious effects on SIP purification at later stages of the procedure. The omission of the chymotrypsin digestion, and the inclusion of gel-filtration chromatography in the final clean-up step, resulted in the purification of SIP comparable with that achieved with affinity chromatography.

Two-Step Deterministic Remote Preparation of an Arbitrary Quantum State

NASA Astrophysics Data System (ADS)

Wang, Mei-Yu; Yan, Feng-Li

2010-11-01

We present a two-step deterministic remote state preparation protocol for an arbitrary quhit with the aid of a three-particle Greenberger—Horne—Zeilinger state. Generalization of this protocol for higher-dimensional Hilbert space systems among three parties is also given. We show that only single-particle von Neumann measurements, local operations, and classical communication are necessary. Moreover, since the overall information of the quantum state can be divided into two different pieces, which may be at different locations, this protocol may be useful in the quantum information field.

Isolation and purification of all-trans diadinoxanthin and all-trans diatoxanthin from diatom Phaeodactylum tricornutum.

PubMed

Kuczynska, Paulina; Jemiola-Rzeminska, Malgorzata

2017-01-01

Two diatom-specific carotenoids are engaged in the diadinoxanthin cycle, an important mechanism which protects these organisms against photoinhibition caused by absorption of excessive light energy. A high-performance and economical procedure of isolation and purification of diadinoxanthin and diatoxanthin from the marine diatom Phaeodactylum tricornutum using a four-step procedure has been developed. It is based on the use of commonly available materials and does not require advanced technology. Extraction of pigments, saponification, separation by partition and then open column chromatography, which comprise the complete experimental procedure, can be performed within 2 days. This method allows HPLC grade diadinoxanthin and diatoxanthin of a purity of 99 % or more to be obtained, and the efficiency was estimated to be 63 % for diadinoxanthin and 73 % for diatoxanthin. Carefully selected diatom culture conditions as well as analytical ones ensure highly reproducible performance. A protocol can be used to isolate and purify the diadinoxanthin cycle pigments both on analytical and preparative scale.

A simple approach for human recombinant apolipoprotein E4 expression and purification.

PubMed

Argyri, Letta; Skamnaki, Vassiliki; Stratikos, Efstratios; Chroni, Angeliki

2011-10-01

We report a simple expression and purification procedure for the production of recombinant apolipoprotein E4 (apoE4), an important protein for the lipid homeostasis in humans that plays critical roles in the pathogenesis of cardiovascular and neurodegenerative diseases. Our approach is based on the expression of a thioredoxin-apoE4 fusion construct in bacterial cells and subsequent removal of the fused thioredoxin using the highly specific 3C protease, avoiding costly and laborious lipidation-delipidation steps used before. Our approach results in rapid, high-yield production of structurally and functionally competent apoE4 as evidenced by secondary structure measurements, thermal and chemical melting profiles and the kinetic profile of solubilization of dimyristoyl-phosphatidylcholine (DMPC) vesicles. This protocol is appropriate for laboratories with little experience in apolipoprotein biochemistry and will facilitate future studies on the role of apoE4 in the pathogenesis of cardiovascular disease and neurodegenerative diseases, including Alzheimer's disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

PubMed Central

Saraswat, Mayank; Ravidá, Alessandra; Holthofer, Harry

2013-01-01

Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. PMID:24455685

Purification of Logic-Qubit Entanglement

PubMed Central

Zhou, Lan; Sheng, Yu-Bo

2016-01-01

Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network. PMID:27377165

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

PubMed

Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

1994-06-01

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories.

PubMed

Hughes, Laura; Wilkins, Kimberly; Goldsmith, Cynthia S; Smith, Scott; Hudson, Paul; Patel, Nishi; Karem, Kevin; Damon, Inger; Li, Yu; Olson, Victoria A; Satheshkumar, P S

2017-05-01

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron ® . Genetron ® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield. Published by Elsevier B.V.

Enriching the biological space of natural products and charting drug metabolites, through real time biotransformation monitoring: The NMR tube bioreactor.

PubMed

Chatzikonstantinou, Alexandra V; Chatziathanasiadou, Maria V; Ravera, Enrico; Fragai, Marco; Parigi, Giacomo; Gerothanassis, Ioannis P; Luchinat, Claudio; Stamatis, Haralambos; Tzakos, Andreas G

2018-01-01

Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Herein, we developed a simple and time-effective process, the "NMR-tube bioreactor", that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets. We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery. This process can speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process.

PubMed

Gea, Saharman; Reynolds, Christopher T; Roohpour, Nima; Wirjosentono, Basuki; Soykeabkaew, Nattakan; Bilotti, Emiliano; Peijs, Ton

2011-10-01

Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5 wt.% NaOH solution produced a twofold increase in Young's modulus of processed BC sheet over untreated BC sheet. Further enhancements are achieved after a second treatment with 2.5 wt.% NaOCl (bleaching). These treatments were carefully designed in order to prevent any polymorphic crystal transformation from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morphological and thermal stability of the processed films are also improved. Copyright © 2011 Elsevier Ltd. All rights reserved.

Polarization entanglement purification for concatenated Greenberger-Horne-Zeilinger state

NASA Astrophysics Data System (ADS)

Zhou, Lan; Sheng, Yu-Bo

2017-10-01

Entanglement purification plays a fundamental role in long-distance quantum communication. In the paper, we put forward the first polarization entanglement purification protocol (EPP) for one type of nonlocal logic-qubit entanglement, i.e., concatenated Greenberger-Horne-Zeilinger (C-GHZ) state, resorting to the photon-atom interaction in low-quality (Q) cavity. In contrast to existing EPPs, this protocol can purify the bit-flip error and phase-flip error in both physic and logic level. Instead of measuring the photons directly, this protocol only requires to measure the atom states to judge whether the protocol is successful. In this way, the purified logic entangled states can be preserved for further application. Moreover, it makes this EPP repeatable so as to obtain a higher fidelity of logic entangled states. As the logic-qubit entanglement utilizes the quantum error correction (QEC) codes, which has an inherent stability against noise and decoherence, this EPP combined with the QEC codes may provide a double protection for the entanglement from the channel noise and may have potential applications in long-distance quantum communication.

A cell culture-derived whole virus influenza A vaccine based on magnetic sulfated cellulose particles confers protection in mice against lethal influenza A virus infection.

PubMed

Pieler, Michael M; Frentzel, Sarah; Bruder, Dunja; Wolff, Michael W; Reichl, Udo

2016-12-07

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening studies requiring fast purification protocols. Copyright © 2016 Elsevier Ltd. All rights reserved.

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

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

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

Application of hydrophobic interaction displacement chromatography for an industrial protein purification.

PubMed

Sunasara, Khurram M; Xia, Fang; Gronke, Robert S; Cramer, Steven M

2003-05-05

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C-terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high-throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary-phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG-3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step-gradient elution process. Copyright 2003 Wiley Periodicals, Inc.

Supercritical CO2 assisted process for the production of high-purity and sterile nano-hydroxyapatite/chitosan hybrid scaffolds.

PubMed

Ruphuy, G; Souto-Lopes, M; Paiva, D; Costa, P; Rodrigues, A E; Monteiro, F J; Salgado, C L; Fernandes, M H; Lopes, J C; Dias, M M; Barreiro, M F

2018-04-01

Hybrid scaffolds composed of hydroxyapatite (HAp), in particular in its nanometric form (n-HAp), and chitosan (CS) are promising materials for non-load-bearing bone graft applications. The main constraints of their production concern the successful implementation of the final purification/neutralization and sterilization steps. Often, the used purification strategies can compromise scaffold structural features, and conventional sterilization techniques can result in material's thermal degradation and/or contamination with toxic residues. In this context, this work presents a process to produce n-HAp/CS scaffolds mimicking bone composition and structure, where an innovative single step based on supercritical CO 2 extraction was used for both purification and sterilization. A removal of 80% of the residual acetic acid was obtained (T = 75°C, p = 8.0 MPa, 2 extraction cycles of 2 h) giving rise to scaffolds exhibiting adequate interconnected porous structure, fast swelling and storage modulus compatible with non-load-bearing applications. Moreover, the obtained scaffolds showed cytocompatibility and osteoconductivity without further need of disinfection/sterilization procedures. Among the main advantages, the proposed process comprises only three steps (n-HAp/CS dispersion preparation; freeze-drying; and supercritical CO 2 extraction), and the supercritical CO 2 extraction show clear advantages over currently used procedures based on neutralization steps. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 965-975, 2018. © 2017 Wiley Periodicals, Inc.

Microwave-Assisted γ-Valerolactone Production for Biomass Lignin Extraction: A Cascade Protocol.

PubMed

Tabasso, Silvia; Grillo, Giorgio; Carnaroglio, Diego; Calcio Gaudino, Emanuela; Cravotto, Giancarlo

2016-03-26

The general need to slow the depletion of fossil resources and reduce carbon footprints has led to tremendous effort being invested in creating "greener" industrial processes and developing alternative means to produce fuels and synthesize platform chemicals. This work aims to design a microwave-assisted cascade process for a full biomass valorisation cycle. GVL (γ-valerolactone), a renewable green solvent, has been used in aqueous acidic solution to achieve complete biomass lignin extraction. After lignin precipitation, the levulinic acid (LA)-rich organic fraction was hydrogenated, which regenerated the starting solvent for further biomass delignification. This process does not requires a purification step because GVL plays the dual role of solvent and product, while the reagent (LA) is a product of biomass delignification. In summary, this bio-refinery approach to lignin extraction is a cascade protocol in which the solvent loss is integrated into the conversion cycle, leading to simplified methods for biomass valorisation.

Titanium(IV) isopropoxide mediated solution phase reductive amination on an automated platform: application in the generation of urea and amide libraries.

PubMed

Bhattacharyya, S; Fan, L; Vo, L; Labadie, J

2000-04-01

Amine libraries and their derivatives are important targets for high throughput synthesis because of their versatility as medicinal agents and agrochemicals. As a part of our efforts towards automated chemical library synthesis, a titanium(IV) isopropoxide mediated solution phase reductive amination protocol was successfully translated to automation on the Trident(TM) library synthesizer of Argonaut Technologies. An array of 24 secondary amines was prepared in high yield and purity from 4 primary amines and 6 carbonyl compounds. These secondary amines were further utilized in a split synthesis to generate libraries of ureas, amides and sulfonamides in solution phase on the Trident(TM). The automated runs included 192 reactions to synthesize 96 ureas in duplicate and 96 reactions to synthesize 48 amides and 48 sulfonamides. A number of polymer-assisted solution phase protocols were employed for parallel work-up and purification of the products in each step.

A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins1

PubMed Central

Eschenfeldt, William H.; Stols, Lucy; Millard, Cynthia Sanville; Joachimiak, Andrzej; Donnelly, Mark I.

2009-01-01

Summary Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here. PMID:18988021

Fast method for the simultaneous quantification of toxic polyphenols applied to the selection of genotypes of yam bean (Pachyrhizus sp.) seeds.

PubMed

Lautié, E; Rozet, E; Hubert, P; Vandelaer, N; Billard, F; Felde, T Zum; Grüneberg, W J; Quetin-Leclercq, J

2013-12-15

The purpose of the research was to develop and validate a rapid quantification method able to screen many samples of yam bean seeds to determine the content of two toxic polyphenols, namely pachyrrhizine and rotenone. The analytical procedure described is based on the use of an internal standard (dihydrorotenone) and is divided in three steps: microwave assisted extraction, purification by solid phase extraction and assay by ultra high performance liquid chromatography (UHPLC). Each step was included in the validation protocol and the accuracy profiles methodology was used to fully validate the method. The method was fully validated between 0.25 mg and 5 mg pachyrrhizin per gram of seeds and between 0.58 mg/g and 4 mg/g for rotenone. More than one hundred samples from different accessions, locations of growth and harvest dates were screened. Pachyrrhizine concentrations ranged from 3.29 mg/g to lower than 0.25 mg/g while rotenone concentrations ranged from 3.53 mg/g to lower than 0.58 mg/g. This screening along with principal component analysis (PCA) and discriminant analysis (DA) analyses allowed the selection of the more interesting genotypes in terms of low concentrations of these two toxic polyphenols. © 2013 Elsevier B.V. All rights reserved.

Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

PubMed

Gruss, Fabian; Hiller, Sebastian; Maier, Timm

2015-01-01

TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

Purification and characterization of paraoxon hydrolase from rat liver.

PubMed Central

Rodrigo, L; Gil, F; Hernandez, A F; Marina, A; Vazquez, J; Pla, A

1997-01-01

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM. PMID:9032442

Proton irradiation of [18O]O2: production of [18F]F2 and [18F]F2 + [18F] OF2.

PubMed

Bishop, A; Satyamurthy, N; Bida, G; Hendry, G; Phelps, M; Barrio, J R

1996-04-01

The production of 18F electrophilic reagents via the 18O(p,n)18F reaction has been investigated in small-volume target bodies made of aluminum, copper, gold-plated copper and nickel, having straight or conical bore shapes. Three irradiation protocols-single-step, two-step and modified two-step-were used for the recovery of the 18F activity. The single-step irradiation protocol was tested in all the target bodies. Based on the single-step performance, aluminum targets were utilized extensively in the investigation of the two-step and modified two-step irradiation protocols. With an 11-MeV cyclotron and using the two-step irradiation protocol, > 1Ci [18F]F2 was recovered reproducibly from an aluminum target body. Probable radical mechanisms for the formation of OF2 and FONO2 (fluorine nitrate) in the single-step and modified two-step targets are proposed based on the amount of ozone generated and the nitrogen impurity present in the target gases, respectively.

The effect of column purification on cDNA indirect labelling for microarrays

PubMed Central

Molas, M Lia; Kiss, John Z

2007-01-01

Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays. PMID:17597522

The effect of column purification on cDNA indirect labelling for microarrays.

PubMed

Molas, M Lia; Kiss, John Z

2007-06-27

The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.

Exploiting the acylating nature of the imide-Ugi intermediate: a straightforward synthesis of tetrahydro-1,4-benzodiazepin-2-ones.

PubMed

Mossetti, Riccardo; Saggiorato, Dèsirèe; Tron, Gian Cesare

2011-12-16

We describe a simple and novel protocol for the synthesis of tetrahydro-1,4-benzodiazepin-2-ones with three points of diversity, exploiting the acylating properties of the recently rediscovered Ugi-imide. The final compounds can be easily prepared in three synthetic steps using a multicomponent reaction, a Staudinger reduction, and an acylative protocol, with good to excellent yields for each synthetic step.

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

PubMed

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Soluble expression, purification and characterization of the full length IS2 Transposase.

PubMed

Lewis, Leslie A; Astatke, Mekbib; Umekubo, Peter T; Alvi, Shaheen; Saby, Robert; Afrose, Jehan

2011-10-27

The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.

Soluble expression, purification and characterization of the full length IS2 Transposase

PubMed Central

2011-01-01

Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Conclusions Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level. PMID:22032517

Atomic entanglement purification and concentration using coherent state input-output process in low-Q cavity QED regime.

PubMed

Cao, Cong; Wang, Chuan; He, Ling-Yan; Zhang, Ru

2013-02-25

We investigate an atomic entanglement purification protocol based on the coherent state input-output process by working in low-Q cavity in the atom-cavity intermediate coupling region. The information of entangled states are encoded in three-level configured single atoms confined in separated one-side optical micro-cavities. Using the coherent state input-output process, we design a two-qubit parity check module (PCM), which allows the quantum nondemolition measurement for the atomic qubits, and show its use for remote parities to distill a high-fidelity atomic entangled ensemble from an initial mixed state ensemble nonlocally. The proposed scheme can further be used for unknown atomic states entanglement concentration. Also by exploiting the PCM, we describe a modified scheme for atomic entanglement concentration by introducing ancillary single atoms. As the coherent state input-output process is robust and scalable in realistic applications, and the detection in the PCM is based on the intensity of outgoing coherent state, the present protocols may be widely used in large-scaled and solid-based quantum repeater and quantum information processing.

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

A simple protocol for Matrix Assisted Laser Desorption Ionization- time of flight-mass spectrometry (MALDI-TOF-MS) analysis of lipids and proteins in single microsamples of paintings.

PubMed

van der Werf, Inez D; Calvano, Cosima D; Palmisano, Francesco; Sabbatini, Luigia

2012-03-09

A simple protocol, based on Bligh-Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated. Finally, the proposed protocol was successfully applied to the study of a late-15th century Italian panel painting allowing the identification of various proteinaceous and lipid sections in organic binders, such as egg yolk, egg white, animal glue, casein, and drying oil. Copyright © 2011 Elsevier B.V. All rights reserved.

Protein purification and analysis: next generation Western blotting techniques.

PubMed

Mishra, Manish; Tiwari, Shuchita; Gomes, Aldrin V

2017-11-01

Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

Economic Methods of Ginger Protease'sextraction and Purification

NASA Astrophysics Data System (ADS)

Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

A Multistep Synthesis Featuring Classic Carbonyl Chemistry for the Advanced Organic Chemistry Laboratory

ERIC Educational Resources Information Center

Duff, David B.; Abbe, Tyler G.; Goess, Brian C.

2012-01-01

A multistep synthesis of 5-isopropyl-1,3-cyclohexanedione is carried out from three commodity chemicals. The sequence involves an aldol condensation, Dieckmann-type annulation, ester hydrolysis, and decarboxylation. No purification is required until after the final step, at which point gravity column chromatography provides the desired product in…

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

PubMed

Aissaoui, Neyssene; Chobert, Jean-Marc; Haertlé, Thomas; Marzouki, M Nejib; Abidi, Ferid

2017-06-01

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20 kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40 °C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

A Generic Protocol for Purifying Disulfide-Bonded Domains and Random Protein Fragments Using Fusion Proteins with SUMO3 and Cleavage by SenP2 Protease.

PubMed

Besir, Hüseyin

2017-01-01

Recombinant expression of heterologous proteins in E. coli is well established for a wide range of proteins, although in many cases, purifying soluble and properly folded proteins remains challenging (Sorensen and Mortensen, J Biotechnol 115:113-128, 2005; Correa and Oppezzo, Methods Mol Biol 1258:27-44, 2015). Proteins that contain disulfide bonds (e.g., cytokines, growth factors) are often particularly difficult to purify in soluble form and still need optimizing of protocols in almost every step of the process (Berkmen, Protein Expr Purif 82:240-251, 2012; de Marco, Microb Cell Fact 11:129, 2012). Expression of disulfide bonded proteins in the periplasm of E. coli is one approach that can help to obtain soluble protein with the correct disulfide bridges forming in the periplasm. This offers the appropriate conditions for disulfide formation although periplasmic expression can also result in low expression levels and incorrect folding of the target protein (Schlapschy and Skerra, Methods Mol Biol 705:211-224, 2011). Generation of specific antibodies often requires a specific antigenic sequence of a protein in order to get an efficient immune response and minimize cross-reactivity of antibodies. Larger proteins like GST (Glutathione-S-transferase) or MBP (maltose binding protein) as solubilizing fusion partners are frequently used to keep antigens soluble and immunize animals. This approach has the disadvantage that the immune response against the fusion partner leads to additional antibodies that need to be separated from the antigen-specific antibodies. For both classes of proteins mentioned above, a protocol has been developed and optimized using the human version of small ubiquitin-like modifier 3 (SUMO3) protein and its corresponding protease SenP2. This chapter describes the experimental steps for expression, purification, refolding, and cleavage that are applicable to both disulfide-bonded proteins with a defined structure and random protein fragments for antibody generation or larger peptides with defined sequence that are difficult express on their own.

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

PubMed Central

2010-01-01

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research. PMID:20180960

Three steps to writing adaptive study protocols in the early phase clinical development of new medicines

PubMed Central

2014-01-01

This article attempts to define terminology and to describe a process for writing adaptive, early phase study protocols which are transparent, self-intuitive and uniform. It provides a step by step guide, giving templates from projects which received regulatory authorisation and were successfully performed in the UK. During adaptive studies evolving data is used to modify the trial design and conduct within the protocol-defined remit. Adaptations within that remit are documented using non-substantial protocol amendments which do not require regulatory or ethical review. This concept is efficient in gathering relevant data in exploratory early phase studies, ethical and time- and cost-effective. PMID:24980283

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

PubMed Central

Uteng, Marianne; Hauge, Håvard Hildeng; Brondz, Ilia; Nissen-Meyer, Jon; Fimland, Gunnar

2002-01-01

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis. PMID:11823243

Smart Hydrogel Particles: Biomarker Harvesting: One-step affinity purification, size exclusion, and protection against degradation

PubMed Central

Luchini, Alessandra; Geho, David H.; Bishop, Barney; Tran, Duy; Xia, Cassandra; Dufour, Robert; Jones, Clint; Espina, Virginia; Patanarut, Alexis; Zhu, Weidong; Ross, Mark; Tessitore, Alessandra; Petricoin, Emanuel; Liotta, Lance A.

2010-01-01

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. PMID:18076201

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

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

Savabi, F.; Geiger, P.J.; Bessman, S.P.

1984-03-01

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphorus-labeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P/sub i/) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P/sub i/ is described. 20 references.

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

One-step purification of nisin A by immunoaffinity chromatography.

PubMed

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-12-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.

Use of artichoke (Cynara scolymus) flower extract as a substitute for bovine rennet in the manufacture of Gouda-type cheese: characterization of aspartic proteases.

PubMed

Llorente, Berta E; Obregón, Walter David; Avilés, Francesc X; Caffini, Néstor O; Vairo-Cavalli, Sandra

2014-09-15

Artichoke (Cynara scolymus L.) flower extract was assayed with the aim of replacing animal rennet in the manufacture of Gouda-type cheeses from bovine milk. Floral extract coagulated milk within a suitable time for use on an industrial scale, while the yield of cheese obtained was equal to that achieved with bovine abomasum. Five proteolytic fractions with milk-clotting activity were isolated in a two-step purification protocol, three belonging to the cardosin group. Cheeses made with C. scolymus proteases must be brined for a longer period (40 h) to prevent overproteolysis and avoid the development of a background flavor. The type of coagulant (bovine or vegetable) had no significant effect on the cheeses' chemical parameters analyzed throughout ripening, and no significant organoleptic differences were detected between those manufactured with C. scolymus or animal rennet. The results indicate that C. scolymus flower extract is suitable for replacing animal rennet in the production of Gouda-type cheeses. Copyright © 2014 Elsevier Ltd. All rights reserved.

Double C-NOT attack and counterattack on `Three-step semi-quantum secure direct communication protocol'

NASA Astrophysics Data System (ADS)

Gu, Jun; Lin, Po-hua; Hwang, Tzonelih

2018-07-01

Recently, Zou and Qiu (Sci China Phys Mech Astron 57:1696-1702, 2014) proposed a three-step semi-quantum secure direct communication protocol allowing a classical participant who does not have a quantum register to securely send his/her secret message to a quantum participant. However, this study points out that an eavesdropper can use the double C-NOT attack to obtain the secret message. To solve this problem, a modification is proposed.

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

A Copper-Sulfate-Based Inorganic Chemistry Laboratory for First-Year University Students That Teaches Basic Operations and Concepts.

ERIC Educational Resources Information Center

Rodriguez, Emilio; Vicente, Miguel Angel

2002-01-01

Presents a 10-hour chemistry experiment using copper sulfate that has three steps: (1) purification of an ore containing copper sulfate and insoluble basic copper sulfates; (2) determination of the number of water molecules in hydrated copper sulfate; and (3) recovery of metallic copper from copper sulfate. (Author/YDS)

Protocol for Initial Purification of Bacteriocin

DTIC Science & Technology

2015-10-01

lysate/extract preparation, column purification, and a desalting . The peptide was tracked throughout the process using a soft agar overlay activity...tris PAGE. It is necessary to desalt those samples for 150-mM and 1-M fractions, by using dialysis or G10 sephadex columns, in order to prevent

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

PubMed

Bereli, Nilay; Ertürk, Gizem; Tümer, M Aşkin; Say, Ridvan; Denizli, Adil

2013-05-01

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.

Preparation of a one-curie 171Tm target for the Detector for Advanced Neutron Capture Experiments (DANCE)

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

Schwantes, Jon M.; Taylor, Wayne A.; Rundberg, Robert S.

2008-05-15

Roughly one curie of 171Tm (t1/2=1.92a) has been produced and purified for the purpose of making a nuclear target for the first measurements of its neutron capture cross section. Target preparation consisted of three key steps: (1) material production; (2) separation and purification; and (3) electrodeposition onto a suitable backing material. Approximately 1.5 mg of the target material (at the time of separation) was produced by irradiating roughly 250 mg of its stable enriched 170Er lanthanide neighbor with neutrons at the ILL reactor in France. This production method resulted in a “difficult-to-separate” 1:167 mixture of near-neighboring lanthanides, Tm and Er.more » Separation and purification was accomplished using high-performance liquid chromatorgraphy (HPLC), with a proprietary cation exchange column (Dionex, CS-3) and alpha-hydroxyisobutyric acid (a-HIB) eluent. This technique yielded a final product of ~95% purity with respect to Tm. A portion (20 ug) of the Tm was electrodeposited on thin Be foil and delivered to the Los Alamos Neutron Science CEnter (LANSCE) for preliminary analysis of its neutron capture cross section using the Detector for Advanced Neutron Capture Experiments (DANCE). This paper discusses the major hurdles associated with the separation and purification step including, scale-up issues related to the use of HPLC for material separation and purification of the target material from a-HIB and 4-(2-pyridylazo)resorcinol (PAR) colorant.« less

One-step purification of nisin A by immunoaffinity chromatography.

PubMed Central

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-01-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method. PMID:9406424

Semi-Preparative Isolation and Purification of Three Tauro-Conjugated Cholic Acids from Pulvis Fellis Suis by HSCCC Coupled with ELSD Detection.

PubMed

He, Jiao; Zhang, Yongmin; Ito, Yoichiro; Sun, Wenji

2011-01-01

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was applied to the separation and purification of three tauro-conjugated cholic acids of taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA) and taurohyocholic acid (THCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The two-phase solvent system composed of chloroform-methanol-water-acetic acid (4:4:2:0.3, v/v/v/v) was selected for the one-step separation where the lower phase was used as the mobile phase in the head to tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 1.5 ml/min and 25°C respectively. From 100 mg of the crude extract, 10.2 mg of TCDCA, 11.8 mg of THDCA and 5.3 mg of THCA were obtained with the purity of 94.6%, 96.5% and 95.4%, respectively. in one step separation The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three tauro-conjugated cholic acids were identified by ESI-MS, (1)H NMR and (13)C NMR.

Preparative isolation and purification of hainanmurpanin, meranzin, and phebalosin from leaves of Murraya exotica L. using supercritical fluid extraction combined with consecutive high-speed countercurrent chromatography.

PubMed

Yan, Rongwei; Shen, Jie; Liu, Xiaojing; Zou, Yong; Xu, Xinjun

2018-05-01

The objective of this study was to develop a consecutive preparation method for the isolation and purification of hainanmurpanin, meranzin, and phebalosin from leaves of Murraya exotica L. The process involved supercritical fluid extraction with CO 2 , solvent extraction, and two-step high-speed countercurrent chromatography. Pressure, temperature, and the volume of entrainer were optimized as 27 MPa, 52°C, and 60 mL by response surface methodology in supercritical fluid extraction with CO 2 , and the yield of the crude extracts was 7.91 g from 100 g of leaves. Subsequently, 80% methanol/water was used to extract and condense the three compounds from the crude extracts, and 4.23 g of methanol/water extracts was obtained. Then, a two-step high-speed countercurrent chromatography procedure was developed for the isolation of the three target compounds from methanol/water extracts, including conventional high-speed countercurrent chromatography for further enrichment and consecutive high-speed countercurrent chromatography for purification. The yield of concentrates from high-speed countercurrent chromatography was 2.50 g from 4.23 g of methanol/water extracts. Finally, the consecutive high-speed countercurrent chromatography produced 103.2 mg of hainanmurpanin, 244.7 mg of meranzin, and 255.4 mg of phebalosin with purities up to 97.66, 99.36, and 98.64%, respectively, from 900 mg of high-speed countercurrent chromatography concentrates in one run of three consecutive sample loadings without exchanging a solvent system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification and characterisation of fatty acid methyl esters in microalgae: Comparison of pretreatment and purification methods.

PubMed

Lage, Sandra; Gentili, Francesco G

2018-06-01

A systematic qualitative and quantitative analysis of fatty acid methyl esters (FAMEs) is crucial for microalgae species selection for biodiesel production. The aim of this study is to identify the best method to assess microalgae FAMEs composition and content. A single-step method, was tested with and without purification steps-that is, separation of lipid classes by thin-layer chromatography (TLC) or solid-phase extraction (SPE). The efficiency of a direct transesterification method was also evaluated. Additionally, the yield of the FAMEs and the profiles of the microalgae samples with different pretreatments (boiled in isopropanol, freezing, oven-dried and freeze-dried) were compared. The application of a purification step after lipid extraction proved to be essential for an accurate FAMEs characterisation. The purification methods, which included TLC and SPE, provided superior results compared to not purifying the samples. Freeze-dried microalgae produced the lowest FAMEs yield. However, FAMEs profiles were generally equivalent among the pretreatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Process for purification of silicon

NASA Technical Reports Server (NTRS)

Rath, H. J.; Sirtl, E.; Pfeiffer, W.

1981-01-01

The purification of metallurgically pure silicon having a silicon content of more than 95% by weight is accomplished by leaching with an acidic solution which substantially does not attack silicon. A mechanical treatment leading to continuous particle size reduction of the granulated silicon to be purified is combined with the chemical purification step.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

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

PubMed

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

2013-07-15

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

Evaluation of accuracy in implant site preparation performed in single- or multi-step drilling procedures.

PubMed

Marheineke, Nadine; Scherer, Uta; Rücker, Martin; von See, Constantin; Rahlf, Björn; Gellrich, Nils-Claudius; Stoetzer, Marcus

2018-06-01

Dental implant failure and insufficient osseointegration are proven results of mechanical and thermal damage during the surgery process. We herein performed a comparative study of a less invasive single-step drilling preparation protocol and a conventional multiple drilling sequence. Accuracy of drilling holes was precisely analyzed and the influence of different levels of expertise of the handlers and additional use of drill template guidance was evaluated. Six experimental groups, deployed in an osseous study model, were representing template-guided and freehanded drilling actions in a stepwise drilling procedure in comparison to a single-drill protocol. Each experimental condition was studied by the drilling actions of respectively three persons without surgical knowledge as well as three highly experienced oral surgeons. Drilling actions were performed and diameters were recorded with a precision measuring instrument. Less experienced operators were able to significantly increase the drilling accuracy using a guiding template, especially when multi-step preparations are performed. Improved accuracy without template guidance was observed when experienced operators were executing single-step versus multi-step technique. Single-step drilling protocols have shown to produce more accurate results than multi-step procedures. The outcome of any protocol can be further improved by use of guiding templates. Operator experience can be a contributing factor. Single-step preparations are less invasive and are promoting osseointegration. Even highly experienced surgeons are achieving higher levels of accuracy by combining this technique with template guidance. Hereby template guidance enables a reduction of hands-on time and side effects during surgery and lead to a more predictable clinical diameter.

An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

PubMed

Fratz-Berilla, Erica J; Ketcham, Stephanie A; Parhiz, Hamideh; Ashraf, Muhammad; Madhavarao, Chikkathur N

2017-12-01

Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme. Published by Elsevier Inc.

Semiconductor grade, solar silicon purification project

NASA Technical Reports Server (NTRS)

Ingle, W. M.; Chaney, R.; Thompson, S.

1977-01-01

The potential for a three step SiF2 polymer transport purification process was examined. The process involves reacting low cost mg silicon with SiF4 to yield SiF2 gas which is condensed to form polymeric (SiF2)x. The polymer is then heated above 400 C to yield Si, SiF4 and higher Si sub n F sub 2n+2 homologues. This report presents and discusses continuing progress on (1) observations on (SiF2)x polymer formation and depolymerization on the small coil, (2) mass balance studies, (3) partial pressures of SiF2 and SiF4, (4) AlF3 mass spectral studies, and (5) material analysis studies.

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells.

PubMed

Kresoja-Rakic, Jelena; Felley-Bosco, Emanuela

2018-04-25

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Experimental purification of two-atom entanglement.

PubMed

Reichle, R; Leibfried, D; Knill, E; Britton, J; Blakestad, R B; Jost, J D; Langer, C; Ozeri, R; Seidelin, S; Wineland, D J

2006-10-19

Entanglement is a necessary resource for quantum applications--entanglement established between quantum systems at different locations enables private communication and quantum teleportation, and facilitates quantum information processing. Distributed entanglement is established by preparing an entangled pair of quantum particles in one location, and transporting one member of the pair to another location. However, decoherence during transport reduces the quality (fidelity) of the entanglement. A protocol to achieve entanglement 'purification' has been proposed to improve the fidelity after transport. This protocol uses separate quantum operations at each location and classical communication to distil high-fidelity entangled pairs from lower-fidelity pairs. Proof-of-principle experiments distilling entangled photon pairs have been carried out. However, these experiments obtained distilled pairs with a low probability of success and required destruction of the entangled pairs, rendering them unavailable for further processing. Here we report efficient and non-destructive entanglement purification with atomic quantum bits. Two noisy entangled pairs were created and distilled into one higher-fidelity pair available for further use. Success probabilities were above 35 per cent. The many applications of entanglement purification make it one of the most important techniques in quantum information processing.

Doing That Thing That Scientists Do: A Discovery-Driven Module on Protein Purification and Characterization for the Undergraduate Biochemistry Laboratory Classroom

ERIC Educational Resources Information Center

Garrett, Teresa A.; Osmundson, Joseph; Isaacson, Marisa; Herrera, Jennifer

2015-01-01

In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module,…

Necessity of purification during bacterial DNA extraction with environmental soils

PubMed Central

Choi, Jung-Hyun

2017-01-01

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content. PMID:28793754

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

High-throughput screening of chromatographic separations: IV. Ion-exchange.

PubMed

Kelley, Brian D; Switzer, Mary; Bastek, Patrick; Kramarczyk, Jack F; Molnar, Kathleen; Yu, Tianning; Coffman, Jon

2008-08-01

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions. (c) 2008 Wiley Periodicals, Inc.

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

PubMed Central

Murphy, Patrick J. M.

2014-01-01

Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes. PMID:25254496

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves.

PubMed

Oztürk, Lokman; Bülbül, Metin; Elmastas, Mahfuz; Ciftçi, Mehmet

2007-01-01

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.

SU-E-T-581: Planning Evaluation of Step-And-Shoot IMRT, RapidArc and Helical TomoTherapy for Hippocampal-Avoidance Whole Brain Radiotherapy (HA-WBRT).

PubMed

Evans, J; Chen, Q; Wuthrick, E; Weldon, M; Rong, Y

2012-06-01

Several planning strategies are available for hippocampal- avoidance whole-brain radiotherapy (HA-WBRT) following RTOG protocol 0933, but have yet to be compared on a common set of patient data. In this inter-institutional investigation, we evaluate three modalities likely to be employed by protocol participants; step-and-shoot IMRT, volumetric modulated arc therapy, and helical tomotherapy. A common set of patients is used for comparison, including credentialing and successfully accrued patients. Eight patient datasets were selected and de-identified prior to planning. Structures were contoured by physicians per protocol using fused MRI datasets. Three plans were generated for each dataset: Philips Pinnacle 9-field non-coplanar IMRT using protocol recommended beam parameters, Varian's RapidArc using two coplanar arcs, and Accuray's TomoTherapy using a 1cm jaw width. With the goal of meeting the compliance criteria outlined in RTOG 0933 (target coverage and dose limits to the hippocampus and optic structures), three planners independently planned each modality without prior knowledge of the patient's other plans to reduce bias. The three plans for each patient were compared according to the protocol's dosimetric compliance criteria. A homogeneity index was also computed to compare target dose uniformity. All plans achieved the protocol dose criteria, except for one RapidArc plan with slightly inferior dose to the optic chiasm. TomoTherapy offered superior dose homogeneity for all patients. For the two linac based methods, RapidArc was found to provide dose homogeneity at least as good as, and in most cases superior to, 9-field step-and-shoot IMRT. Helical TomoTherapy offers superior dose homogeneity for HA-WBRT following RTOG 0933. Compared to step-and-shoot IMRT, volumetric modulated arc techniques, such as RapidArc, can offer improved homogeneity for HA- WBRT and are generally more efficient/expeditious to deliver than the noncoplanar 9-field arrangement recommended by the protocol, which uses 7 separate couch angles. © 2012 American Association of Physicists in Medicine.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

PubMed

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Isolation of Plant Photosystem II Complexes by Fractional Solubilization

PubMed Central

Haniewicz, Patrycja; Floris, Davide; Farci, Domenica; Kirkpatrick, Joanna; Loi, Maria C.; Büchel, Claudia; Bochtler, Matthias; Piano, Dario

2015-01-01

Photosystem II (PSII) occurs in different forms and supercomplexes in thylakoid membranes. Using a transplastomic strain of Nicotiana tabacum histidine tagged on the subunit PsbE, we have previously shown that a mild extraction protocol with β-dodecylmaltoside enriches PSII characteristic of lamellae and grana margins. Here, we characterize residual granal PSII that is not extracted by this first solubilization step. Using affinity purification, we demonstrate that this PSII fraction consists of PSII-LHCII mega- and supercomplexes, PSII dimers, and PSII monomers, which were separated by gel filtration and functionally characterized. Our findings represent an alternative demonstration of different PSII populations in thylakoid membranes, and they make it possible to prepare PSII-LHCII supercomplexes in high yield. PMID:26697050

Tall fescue seed extraction and partial purification of ergot alkaloids

PubMed Central

Ji, Huihua; Fannin, F.; Klotz, J.; Bush, Lowell

2014-01-01

Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline. PMID:25566528

Tall fescue seed extraction and partial purification of ergot alkaloids

NASA Astrophysics Data System (ADS)

Bush, Lowell

2014-12-01

Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W×L×D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature. Resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v) and the hexane fraction was discarded. The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

PubMed Central

Xiao, Rong; Anderson, Stephen; Aramini, James; Belote, Rachel; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John K.; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Jiang, Mei; Kornhaber, Gregory J.; Lee, Dong Yup; Locke, Jessica Y.; Ma, Li-Chung; Maglaqui, Melissa; Mao, Lei; Mitra, Saheli; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Sharma, Seema; Shastry, Ritu; Swapna, G.V.T.; Tong, Saichu N.; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.; Acton, Thomas B.

2014-01-01

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities. PMID:20688167

Peptidome characterization and bioactivity analysis of donkey milk.

PubMed

Piovesana, Susy; Capriotti, Anna Laura; Cavaliere, Chiara; La Barbera, Giorgia; Samperi, Roberto; Zenezini Chiozzi, Riccardo; Laganà, Aldo

2015-04-24

Donkey milk is an interesting commercial product for its nutritional values, which make it the most suitable mammalian milk for human consumption, and for the bioactivity associated with it and derivative products. To further mine the characterization of donkey milk, an extensive peptidomic study was performed. Two peptide purification strategies were compared to remove native proteins and lipids and enrich the peptide fraction. In one case the whole protein content was precipitated by organic solvent using cold acetone. In the other one the precipitation of the most abundant milk proteins, caseins, was performed under acidic conditions by acetic acid at pH4.6, instead. The procedures were compared and proved to be partially complementary. Considered together they provided 1330 peptide identifications for donkey milk, mainly coming from the most abundant proteins in milk. The bioactivity of the isolated peptides was also investigated, both by angiotensin-converting-enzyme inhibitory and antioxidant activity assays and by bioinformatics, proving that the isolated peptides did have the tested biological activities. The rationale behind this study is that peptides in food matrices often play an important biological role and, despite the extensive study of the protein composition of different samples, they remain poorly characterized. In fact, in a typical shotgun proteomics study endogenous peptides are not properly characterized. In proteomics workflows one limiting point is the isolation process: if it is specific for the purification of proteins, it often comprises a precipitation step which aims at isolating pure protein pellets and remove unwonted interferent compounds. In this way endogenous peptides, which are not effectively precipitated as well as proteins, are removed too and not analyzed at the end of the process. Moreover, endogenous peptides do often originate from precursor proteins, but in phenomena which are independent of the shotgun digestion protocol, thus they can be obtained from cleavage specificities other than trypsin's, which is the main proteolytic enzyme employed in proteomic experiments. For this reason, in the end, database search will not be effective for identification of these peptides, thus the need to provide different workflows for peptide analysis. In the work presented in this paper this issue is considered for the first time for the analysis of the peptides isolated in donkey milk samples, which have been chosen for its nutritional interest. This study provides additional knowledge on this milk, already characterized by traditional proteomics studies and peptidomic studies after simulated digestion. This type of study is not just a description of the naturally occurring peptidome of a sample, but also represents a starting point to discover and characterize those naturally occurring peptides responsible for the observed bioactivities of biological samples, as in the case of donkey milk, which would remain uncharacterized by other approaches. In this paper an analytical protocol was described for the efficient isolation and purification of peptides in donkey milk, assessing the effect of the purification protocol on the final identifications. Purified peptide samples were also checked to empirically elucidate any ACE inhibitory or antioxidant activity. Finally, the peptidomic results were also further mined by a bioinformatic-driven approach for bioactive peptide identification in the donkey milk samples. In our opinion, the main strengths of this study are related to the improved analytical workflow (either as purification protocol comparison or analytical platform development) which provides a high number of identified peptides, for which the biological significance as potential bioactive peptides has also been investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

Scaled-up production of poacic acid, a plant-derived antifungal agent

DOE PAGES

Yue, Fengxia; Gao, Ruili; Piotrowski, Jeff S.; ...

2017-09-01

Poacic acid, a decarboxylated product from 8–5-diferulic acid that is commonly found in monocot lignocellulosic hydrolysates, has been identified as a natural antifungal agent against economically significant fungi and oomycete plant pathogens. Starting from commercially available or monocot-derivable ferulic acid, a three-step synthetic procedure has been developed for the production of poacic acid needed for field testing in a controlled agricultural setting. First, ferulic acid was esterified to produce ethyl ferulate in 92% yield. Second, peroxidase-catalyzed free radical dehydrodimerization of ethyl ferulate produced crude diferulates, mainly 8–5-diferulate, in 91% yield. Finally, crystalline poacic acid was obtained in 25% yield viamore » alkaline hydrolysis of the crude diferulates after purification by flash-column chromatography. Thus, this new procedure offers two key improvements relevant to large-scale production: 1) bubbling air through the reaction mixture in the second step to remove acetone greatly improves the recovery efficiency of the crude diferulates; and 2) telescoping minor impurities directly into the alkaline hydrolysis step eliminates the need for additional column purifications, thus reducing the overall cost of production and removing a major impediment to process scale-up.« less

Scaled-up production of poacic acid, a plant-derived antifungal agent

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

Yue, Fengxia; Gao, Ruili; Piotrowski, Jeff S.

Poacic acid, a decarboxylated product from 8–5-diferulic acid that is commonly found in monocot lignocellulosic hydrolysates, has been identified as a natural antifungal agent against economically significant fungi and oomycete plant pathogens. Starting from commercially available or monocot-derivable ferulic acid, a three-step synthetic procedure has been developed for the production of poacic acid needed for field testing in a controlled agricultural setting. First, ferulic acid was esterified to produce ethyl ferulate in 92% yield. Second, peroxidase-catalyzed free radical dehydrodimerization of ethyl ferulate produced crude diferulates, mainly 8–5-diferulate, in 91% yield. Finally, crystalline poacic acid was obtained in 25% yield viamore » alkaline hydrolysis of the crude diferulates after purification by flash-column chromatography. Thus, this new procedure offers two key improvements relevant to large-scale production: 1) bubbling air through the reaction mixture in the second step to remove acetone greatly improves the recovery efficiency of the crude diferulates; and 2) telescoping minor impurities directly into the alkaline hydrolysis step eliminates the need for additional column purifications, thus reducing the overall cost of production and removing a major impediment to process scale-up.« less

Purification of Hemoglobin by Tangential Flow Filtration with Diafiltration

PubMed Central

Elmer, Jacob; Harris, David R.; Sun, Guoyong; Palmer, Andre F.

2009-01-01

A recent study by Palmer et al. (2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) & 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three-stage TFF process is sufficient to produce HPLC-grade bHb. PMID:19621471

Phage display of engineered binding proteins.

PubMed

Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

2014-01-01

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Using an intervention mapping approach to develop a discharge protocol for intensive care patients.

PubMed

van Mol, Margo; Nijkamp, Marjan; Markham, Christine; Ista, Erwin

2017-12-19

Admission into an intensive care unit (ICU) may result in long-term physical, cognitive, and emotional consequences for patients and their relatives. The care of the critically ill patient does not end upon ICU discharge; therefore, integrated and ongoing care during and after transition to the follow-up ward is pivotal. This study described the development of an intervention that responds to this need. Intervention Mapping (IM), a six-step theory- and evidence-based approach, was used to guide intervention development. The first step, a problem analysis, comprised a literature review, six semi-structured telephone interviews with former ICU-patients and their relatives, and seven qualitative roundtable meetings for all eligible nurses (i.e., 135 specialized and 105 general ward nurses). Performance and change objectives were formulated in step two. In step three, theory-based methods and practical applications were selected and directed at the desired behaviors and the identified barriers. Step four designed a revised discharge protocol taking into account existing interventions. Adoption, implementation and evaluation of the new discharge protocol (IM steps five and six) are in progress and were not included in this study. Four former ICU patients and two relatives underlined the importance of the need for effective discharge information and supportive written material. They also reported a lack of knowledge regarding the consequences of ICU admission. 42 ICU and 19 general ward nurses identified benefits and barriers regarding discharge procedures using three vignettes framed by literature. Some discrepancies were found. For example, ICU nurses were skeptical about the impact of writing a lay summary despite extensive evidence of the known benefits for the patients. ICU nurses anticipated having insufficient skills, not knowing the patient well enough, and fearing legal consequences of their writings. The intervention was designed to target the knowledge, attitudes, self-efficacy, and perceived social influence. Building upon IM steps one to three, a concept discharge protocol was developed that is relevant and feasible within current daily practice. Intervention mapping provided a comprehensive framework to improve ICU discharge by guiding the development process of a theory- and empirically-based discharge protocol that is robust and useful in practice.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

PubMed

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Mathematical modeling of the whole expanded bed adsorption process to recover and purify chitosanases from the unclarified fermentation broth of Paenibacillus ehimensis.

PubMed

de Araújo Padilha, Carlos Eduardo; Fortunato Dantas, Paulo Victor; de Sousa, Francisco Canindé; de Santana Souza, Domingos Fabiano; de Oliveira, Jackson Araújo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

2016-12-15

In this study, a general rate model was applied to the entire process of expanded bed adsorption chromatography (EBAC) for the chitosanases purification protocol from unclarified fermentation broth produced by Paenibacillus ehimensis using the anionic adsorbent Streamline ® DEAE. For the experiments performed using the expanded bed, a homemade column (2.6cm×30.0cm) was specially designed. The proposed model predicted the entire EBA process adequately, giving R 2 values higher than 0.85 and χ 2 as low as 0.351 for the elution step. Using the validated model, a 3 3 factorial design was used to investigate other non-tested conditions as input. It was observed that the superficial velocity during loading and washing steps, as well as the settled bed height, has a strong positive effect on the F objective function used to evaluate the production of the purified chitosanases. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Cloning, expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from Leishmania braziliensis (LbCYP51).

PubMed

Freitas, Humberto F; Leal Pires, Acássia Benjamim; Castilho, Marcelo S

2018-04-01

Leishmaniasis, a neglected tropical disease, is a major cause of morbidity and mortality worldwide. Of the three main clinical forms, cutaneous leishmaniasis (CL) is the most common and 40 million people are at risk in the endemic areas. Currently, the available drugs to fight leishmaniasis have high toxicity and poor efficiency. Then, it is very important to search for effective and safe drugs that would target essential enzymes from the parasite, such as lanosterol 14-alpha demethylase (CYP51, EC 1.14.13.70) from Leishmania braziliensis. Because most drug design efforts have been directed for Leishmania non-braziliensis species, there is no structural or kinetic data regarding L. braziliensis CYP51. Herein, we present for the first time molecular biology efforts and purification protocol to obtain the enzyme LbCYP51. These results lay the ground for future investigation of drugs against this target.

Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis.

PubMed

Geisbrecht, Brian V; Nikonenko, Boris; Samala, Rowena; Nakamura, Reiko; Nacy, Carol A; Sacksteder, Katherine A

2006-03-01

Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.

Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

PubMed

Verlotta, Angelo; Trono, Daniela

2014-09-01

Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature. Copyright © 2014 Elsevier Inc. All rights reserved.

Quadrupole Magnetic Sorting of Porcine Islets of Langerhans

PubMed Central

Shenkman, Rustin M.; Chalmers, Jeffrey J.; Hering, Bernhard J.; Kirchhof, Nicole

2009-01-01

Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. Inconsistent isolation, purification, and recovery of large numbers of high-quality islets remain substantial impediments to progress in the field. Removing islets as soon as they are liberated from the pancreas during digestion and circumventing the need for density gradient purification is likely to result in substantially increased viable islet yields by minimizing exposure to proteolytic enzymes, reactive oxygen intermediates, and mechanical stress associated with centrifugation. This study capitalized on the hypervascularity of islets compared with acinar tissue to explore their preferential enrichment with magnetic beads to enable immediate separation in a magnetic field utilizing a quadrupole magnetic sorting. The results demonstrate that (1) preferential enrichment of porcine islets is achievable, but homogeneous bead distribution within the pancreas is difficult to achieve with current protocols; (2) greater than 70% of islets in the dissociated pancreatic tissue were recovered by quadrupole magnetic sorting, but their purity was low; and (3) infused islets purified by density gradients and subsequently passed through quadrupole magnetic sorting had similar potency as uninfused islets. These results demonstrate proof of concept and define the steps for implementation of this technology in pig and human islet isolation. PMID:19505179

Carbon Nanotube Activities at NASA-Johnson Space Center

NASA Technical Reports Server (NTRS)

Arepalli, Sivaram

2006-01-01

Research activities on carbon nanotubes at NASA-Johnson Space Center include production, purification, characterization and their applications for human space flight. In-situ diagnostics during nanotube production by laser oven process include collection of spatial and temporal data of passive emission and laser induced fluorescence from C2, C3 and Nickel atoms in the plume. Details of the results from the "parametric study" of the pulsed laser ablation process indicate the effect of production parameters including temperature, buffer gas, flow rate, pressure, and laser fluence. Improvement of the purity by a variety of steps in the purification process is monitored by characterization techniques including SEM, TEM, Raman, UV-VIS-NIR and TGA. A recently established NASA-JSC protocol for SWCNT characterization is undergoing revision with feedback from nanotube community. Efforts at JSC over the past five years in composites have centered on structural polymednanotube systems. Recent activities broadened this focus to multifunctional materials, supercapacitors, fuel cells, regenerable CO2 absorbers, electromagnetic shielding, radiation dosimetry and thermal management systems of interest for human space flight. Preliminary tests indicate improvement of performance in most of these applications because of the large surface area as well as high electrical and thermal conductivity exhibited by SWCNTs.

Selective Precipitation and Purification of Monovalent Proteins Using Oligovalent Ligands and Ammonium Sulfate

PubMed Central

Mirica, Katherine A.; Lockett, Matthew R.; Snyder, Phillip W.; Shapiro, Nathan D.; Mack, Eric T.; Nam, Sarah; Whitesides, George M.

2012-01-01

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: i) the removal of high-molecular weight impurities through the addition of ammonium sulfate to the crude cell lysate; ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins—for which appropriate oligovalent ligands can be synthesized—and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation. PMID:22188202

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate.

PubMed

Mirica, Katherine A; Lockett, Matthew R; Snyder, Phillip W; Shapiro, Nathan D; Mack, Eric T; Nam, Sarah; Whitesides, George M

2012-02-15

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.

Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.

PubMed

Smejkal, Benjamin; Agrawal, Neeraj J; Helk, Bernhard; Schulz, Henk; Giffard, Marion; Mechelke, Matthias; Ortner, Franziska; Heckmeier, Philipp; Trout, Bernhardt L; Hekmat, Dariusch

2013-09-01

The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies. Copyright © 2013 Wiley Periodicals, Inc.

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

PubMed Central

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

2018-01-01

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

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

PubMed

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

2018-01-01

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

Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

PubMed Central

Zang, Yuguo; Kammerer, Bernd; Eisenkolb, Maike; Lohr, Katrin; Kiefer, Hans

2011-01-01

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step. PMID:21966480

A flow-through chromatography process for influenza A and B virus purification.

PubMed

Weigel, Thomas; Solomaier, Thomas; Peuker, Alessa; Pathapati, Trinath; Wolff, Michael W; Reichl, Udo

2014-10-01

Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines. Copyright © 2014 Elsevier B.V. All rights reserved.

Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale.

PubMed

Schirmer, Emily B; Golden, Kathryn; Xu, Jin; Milling, Jesse; Murillo, Alec; Lowden, Patricia; Mulagapati, Srihariraju; Hou, Jinzhao; Kovalchin, Joseph T; Masci, Allyson; Collins, Kathryn; Zarbis-Papastoitsis, Gregory

2013-08-01

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

PubMed

Masterson, Larry R; Bortone, Nadia; Yu, Tao; Ha, Kim N; Gaffarogullari, Ece C; Nguyen, Oanh; Veglia, Gianluigi

2009-04-01

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.

Distillation and purification of symmetric entangled Gaussian states

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

Fiurasek, Jaromir

2010-10-15

We propose an entanglement distillation and purification scheme for symmetric two-mode entangled Gaussian states that allows to asymptotically extract a pure entangled Gaussian state from any input entangled symmetric Gaussian state. The proposed scheme is a modified and extended version of the entanglement distillation protocol originally developed by Browne et al. [Phys. Rev. A 67, 062320 (2003)]. A key feature of the present protocol is that it utilizes a two-copy degaussification procedure that involves a Mach-Zehnder interferometer with single-mode non-Gaussian filters inserted in its two arms. The required non-Gaussian filtering operations can be implemented by coherently combining two sequences ofmore » single-photon addition and subtraction operations.« less

A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrisanthemi ligand-gated ion channel

PubMed Central

Elberson, Benjamin W.; Whisenant, Ty E.; Cortes, D. Marien; Cuello, Luis G.

2017-01-01

The Erwinia chrisanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC’s function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol. PMID:28279818

An improved 96-well turbidity assay for T4 lysozyme activity.

PubMed

Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

An improved 96-well turbidity assay for T4 lysozyme activity

PubMed Central

Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

Label-Free Biomarker Detection from Whole Blood

DTIC Science & Technology

2010-02-01

we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip...nanosensors to purify biomarkers of interest. This microfluidic purification chip (MPC) captures cancer biomarkers from physiological solutions and, after...assay validation experiments (Fig. 2c). As shown in Fig. 1d, after a second valve switching step transfers MPC contents to the nanosen- sor chip, the

Heat and mass transfer during the cryopreservation of a bioartificial liver device: a computational model.

PubMed

Balasubramanian, Saravana K; Coger, Robin N

2005-01-01

Bioartificial liver devices (BALs) have proven to be an effective bridge to transplantation for cases of acute liver failure. Enabling the long-term storage of these devices using a method such as cryopreservation will ensure their easy off the shelf availability. To date, cryopreservation of liver cells has been attempted for both single cells and sandwich cultures. This study presents the potential of using computational modeling to help develop a cryopreservation protocol for storing the three dimensional BAL: Hepatassist. The focus is upon determining the thermal and concentration profiles as the BAL is cooled from 37 degrees C-100 degrees C, and is completed in two steps: a cryoprotectant loading step and a phase change step. The results indicate that, for the loading step, mass transfer controls the duration of the protocol, whereas for the phase change step, when mass transfer is assumed negligible, the latent heat released during freezing is the control factor. The cryoprotocol that is ultimately proposed considers time, cooling rate, and the temperature gradients that the cellular space is exposed to during cooling. To our knowledge, this study is the first reported effort toward designing an effective protocol for the cryopreservation of a three-dimensional BAL device.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

PubMed

Kelley, Brian

2007-01-01

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

Genetic transformation of a clinical (genital tract), plasmid-free isolate of Chlamydia trachomatis: engineering the plasmid as a cloning vector.

PubMed

Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

2013-01-01

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

Development of Purification Protocol Specific for Bacteriocin 105B

DTIC Science & Technology

2017-02-09

determined to exhibit activity against a pathogenic organism of interest to the Army, Bacillus anthracis Sterne, a surrogate of the active form of...employment in future assays and development into a platform, such as a textile, that relays antimicrobial activity . 15. SUBJECT TERMS 105B...Purification with Ion Exchange Column Chromatography. Representative activity drop test assay evaluating activity of fractions collected from ion

Pathogen vacuole purification from legionella-infected amoeba and macrophages.

PubMed

Hoffmann, Christine; Finsel, Ivo; Hilbi, Hubert

2013-01-01

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.

Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

PubMed

Mirahmadi-Zare, Seyede Zohreh; Allafchian, Alireza; Aboutalebi, Fatemeh; Shojaei, Pendar; Khazaie, Yahya; Dormiani, Kianoush; Lachinani, Liana; Nasr-Esfahani, Mohammad-Hossein

2016-05-01

Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 μg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding. Copyright © 2016 Elsevier Inc. All rights reserved.

Performances of different protocols for exocellular polysaccharides extraction from milk acid gels: Application to yogurt.

PubMed

Nguyen, An Thi-Binh; Nigen, Michaël; Jimenez, Luciana; Ait-Abderrahim, Hassina; Marchesseau, Sylvie; Picart-Palmade, Laetitia

2018-01-15

Dextran or xanthan were used as model exocellular polysaccharides (EPS) to compare the extraction efficiency of EPS from skim milk acid gels using three different protocols. Extraction yields, residual protein concentrations and the macromolecular properties of extracted EPS were determined. For both model EPS, the highest extraction yield (∼80%) was obtained when samples were heated in acidic conditions at the first step of extraction (Protocol 1). Protocols that contained steps of acid/ethanol precipitation without heating (Protocols 2 and 3) show lower extraction yields (∼55%) but allow a better preservation of the EPS macromolecular properties. Changing the pH of acid gels up to 7 before extraction (Protocol 3) improved the extraction yield of anionic EPS without effect on the macromolecular properties of EPS. Protocol 1 was then applied for the quantification of EPS produced during the yogurt fermentation, while Protocol 3 was dedicated to their macromolecular characterization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Butelase-mediated cyclization and ligation of peptides and proteins.

PubMed

Nguyen, Giang K T; Qiu, Yibo; Cao, Yuan; Hemu, Xinya; Liu, Chuan-Fa; Tam, James P

2016-10-01

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Antibiotics from Pseudomonas reptilivora II. Isolation, Purification, and Properties1

PubMed Central

Del Rio, Luís A.; Gorgé, J. López; Olivares, J.; Mayor, F.

1972-01-01

Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B1, and B2. The B1 and B2 activities were studied in greater detail than A. The B1 substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively. Images PMID:4790558

Three-step semiquantum secure direct communication protocol

NASA Astrophysics Data System (ADS)

Zou, XiangFu; Qiu, DaoWen

2014-09-01

Quantum secure direct communication is the direct communication of secret messages without need for establishing a shared secret key first. In the existing schemes, quantum secure direct communication is possible only when both parties are quantum. In this paper, we construct a three-step semiquantum secure direct communication (SQSDC) protocol based on single photon sources in which the sender Alice is classical. In a semiquantum protocol, a person is termed classical if he (she) can measure, prepare and send quantum states only with the fixed orthogonal quantum basis {|0>, |1>}. The security of the proposed SQSDC protocol is guaranteed by the complete robustness of semiquantum key distribution protocols and the unconditional security of classical one-time pad encryption. Therefore, the proposed SQSDC protocol is also completely robust. Complete robustness indicates that nonzero information acquired by an eavesdropper Eve on the secret message implies the nonzero probability that the legitimate participants can find errors on the bits tested by this protocol. In the proposed protocol, we suggest a method to check Eves disturbing in the doves returning phase such that Alice does not need to announce publicly any position or their coded bits value after the photons transmission is completed. Moreover, the proposed SQSDC protocol can be implemented with the existing techniques. Compared with many quantum secure direct communication protocols, the proposed SQSDC protocol has two merits: firstly the sender only needs classical capabilities; secondly to check Eves disturbing after the transmission of quantum states, no additional classical information is needed.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira.

PubMed

Hauk, P; Carvalho, E; Ho, P L

2011-04-01

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Multipinhole SPECT helical scan parameters and imaging volume

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

Yao, Rutao, E-mail: rutaoyao@buffalo.edu; Deng, Xiao; Wei, Qingyang

Purpose: The authors developed SPECT imaging capability on an animal PET scanner using a multiple-pinhole collimator and step-and-shoot helical data acquisition protocols. The objective of this work was to determine the preferred helical scan parameters, i.e., the angular and axial step sizes, and the imaging volume, that provide optimal imaging performance. Methods: The authors studied nine helical scan protocols formed by permuting three rotational and three axial step sizes. These step sizes were chosen around the reference values analytically calculated from the estimated spatial resolution of the SPECT system and the Nyquist sampling theorem. The nine helical protocols were evaluatedmore » by two figures-of-merit: the sampling completeness percentage (SCP) and the root-mean-square (RMS) resolution. SCP was an analytically calculated numerical index based on projection sampling. RMS resolution was derived from the reconstructed images of a sphere-grid phantom. Results: The RMS resolution results show that (1) the start and end pinhole planes of the helical scheme determine the axial extent of the effective field of view (EFOV), and (2) the diameter of the transverse EFOV is adequately calculated from the geometry of the pinhole opening, since the peripheral region beyond EFOV would introduce projection multiplexing and consequent effects. The RMS resolution results of the nine helical scan schemes show optimal resolution is achieved when the axial step size is the half, and the angular step size is about twice the corresponding values derived from the Nyquist theorem. The SCP results agree in general with that of RMS resolution but are less critical in assessing the effects of helical parameters and EFOV. Conclusions: The authors quantitatively validated the effective FOV of multiple pinhole helical scan protocols and proposed a simple method to calculate optimal helical scan parameters.« less

Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein.

PubMed

Chakroun, Maissa; Bel, Yolanda; Caccia, Silvia; Abdelkefi-Mesrati, Lobna; Escriche, Baltasar; Ferré, Juan

2012-07-01

The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae remaining in the first instar), measured at 7d, were similar or even lower than the LC(50) values of mortality at 10d. This strong growth inhibition was not observed when testing the trypsin-activated protein (62 kDa) in either species. S. exigua was less susceptible than S. frugiperda to the protoxin form, with LC(50) values around 10-fold higher. However, both species were equally susceptible to the trypsin-activated form. Processing of Vip3Aa protoxin to the activated form was faster with S. frugiperda midgut juice than with S. exigua midgut juice. The results strongly suggest that the differences in the rate of activation of the Vip3Aa protoxin between both species are the basis for the differences in susceptibility towards the protoxin form. Copyright © 2012 Elsevier Inc. All rights reserved.

CAD/CAM guided surgery in implant dentistry. A review of software packages and step-by-step protocols for planning surgical guides.

PubMed

Scherer, Michael D; Kattadiyil, Mathew T; Parciak, Ewa; Puri, Shweta

2014-01-01

Three-dimensional radiographic imaging for dental implant treatment planning is gaining widespread interest and popularity. However, application of the data from 30 imaging can be a complex and daunting process initially. The purpose of this article is to describe features of three software packages and the respective computerized guided surgical templates (GST) fabricated from them. A step-by-step method of interpreting and ordering a GST to simplify the process of the surgical planning and implant placement is discussed.

Life Cycle Evolution and Systematics of Campanulariid Hydrozoans

DTIC Science & Technology

2004-09-01

kit according to manufacturer’s protocol. Purified PCR product was cycle-sequenced using either Big Dye 2 or 3 sequencing chemistry (ABI), following...ethidium bromide and purified with PCR purification kits (Qiagen). Purified products were cycle- sequenced with either Big Dye 2 or 3 sequencing chemistry...PCR purification kit (Qiagen). The purified product was cycle-sequenced using Big Dye 2 sequencing chemistry (ABI) following the manufacturer’s

Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper

PubMed Central

Smith, LM; Burgoyne, LA

2004-01-01

Background Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. Results FTA® databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA® databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA® paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. Conclusions FTA® paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler. PMID:15072582

On-chip purification and detection of hepatitis C virus RNA from human plasma.

PubMed

Vaghi, V; Potrich, C; Pasquardini, L; Lunelli, L; Vanzetti, L; Ebranati, E; Lai, A; Zehender, G; Mombello, D; Cocuzza, M; Pirri, C F; Pederzolli, C

2016-01-01

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings. Copyright © 2015 Elsevier B.V. All rights reserved.

Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

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

Elkin, Christopher; Kapur, Hitesh; Smith, Troy

2001-09-15

We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620more » phred20 bases as part of Joint Genome Institutes Production Process.« less

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

PubMed Central

Koehl, Ulrike; Brehm, Claudia; Huenecke, Sabine; Zimmermann, Stefanie-Yvonne; Kloess, Stephan; Bremm, Melanie; Ullrich, Evelyn; Soerensen, Jan; Quaiser, Andrea; Erben, Stephanie; Wunram, Claudia; Gardlowski, Tanja; Auth, Eileen; Tonn, Torsten; Seidl, Christian; Meyer-Monard, Sandrine; Stern, Martin; Passweg, Jakob; Klingebiel, Thomas; Bader, Peter; Schwabe, Dirk; Esser, Ruth

2013-01-01

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized. PMID:23730623

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

PubMed

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Inclusion bodies and purification of proteins in biologically active forms.

PubMed

Mukhopadhyay, A

1997-01-01

Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.

Automated multi-dimensional purification of tagged proteins.

PubMed

Sigrell, Jill A; Eklund, Pär; Galin, Markus; Hedkvist, Lotta; Liljedahl, Pia; Johansson, Christine Markeland; Pless, Thomas; Torstenson, Karin

2003-01-01

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

Automated large-scale purification of a G protein-coupled receptor for neurotensin.

PubMed

White, Jim F; Trinh, Loc B; Shiloach, Joseph; Grisshammer, Reinhard

2004-04-30

Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

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

PubMed Central

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

2012-01-01

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

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Standard Free Droplet Digital Polymerase Chain Reaction as a New Tool for the Quality Control of High-Capacity Adenoviral Vectors in Small-Scale Preparations

PubMed Central

Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E.

2015-01-01

Abstract High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

PubMed

Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

2011-11-01

Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps.

PubMed

Roberts, Peter L

2014-01-01

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG-1 & -3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion-exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non-enveloped viruses over the life-time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion-exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X-100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers.

Recovery and purification of chitosanase produced by Bacillus cereus using expanded bed adsorption and central composite design.

PubMed

de Araújo, Nathália Kelly; Pimentel, Vanessa Carvalho; da Silva, Nayane Macedo Portela; de Araújo Padilha, Carlos Eduardo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

2016-02-01

This study presents a system for expanded bed adsorption for the purification of chitosanase from broth extract in a single step. A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01 and used to produce chitosanases. The expanded bed adsorption conditions for chitosanase purification were optimized statistically using STREAMLINE(TM) DEAE and a homemade column (2.6 × 30.0 cm). Dependent variables were defined by the quality criteria purification factor (P) and enzyme yield to optimize the chromatographic process. Statistical analyses showed that the optimum conditions for the maximum P were 150 cm/h load flow velocity, 6.0 cm settled bed height, and 7.36 cm distributor height. Distributor height had a strong influence on the process, considerably affecting both the P and enzyme yield. Optimizing the purification variables resulted in an approximately 3.66-fold increase in the P compared with the value under nonoptimized conditions. This system is promising for the recovery of chitosanase from B. cereus C-01 and is economically viable because it promotes the reduction steps. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

PubMed

Acero-Navarro, Kevin E; Jiménez-Ramírez, Mariella; Villalobos, Miguel A; Vargas-Martínez, Rocío; Perales-Vela, Hugo V; Velasco-García, Roberto

2018-02-01

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl 2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Integration of Gas Chromatography Mass Spectrometry Methods for Differentiating Ricin Preparation Methods

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

Wunschel, David S.; Melville, Angela M.; Ehrhardt, Christopher J.

2012-05-17

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of the castor plant Ricinus communis. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatographicmore » - mass spectrometric (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method and independent of the seed source. In particular the abundance of mannose, arabinose, fucose, ricinoleic acid and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation.« less

Purification and characterization of a hexanol-degrading enzyme extracted from apple

USDA-ARS?s Scientific Manuscript database

An enzyme having activity towards n-hexanol was purified from apple and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 column. The obtained enzyme had a yi...

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification.

PubMed

Koziel, David; Michaelis, Uwe; Kruse, Tobias

2018-08-01

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 10 3 to 10 6 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale. Copyright © 2018. Published by Elsevier B.V.

Purification of a crystallin domain of Yersinia crystallin from inclusion bodies and its comparison to native protein from the soluble fraction.

PubMed

Jobby, M K; Sharma, Yogendra

2006-09-01

It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies. Copyright 2006 John Wiley & Sons, Ltd.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

PubMed Central

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-01-01

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner. PMID:26709825

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

PubMed

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-12-19

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Nanotube Activities at NASA-Johnson Space Center

NASA Technical Reports Server (NTRS)

Arepalli, Sivaram

2004-01-01

Nanotube activities at NASA-Johnson Space Center include production, purification, characterization as well as applications of single wall carbon nanotubes (SWCNTs). A parametric study of the pulsed laser ablation process is recently completed to monitor the effect of production parameters including temperature, buffer gas, flow rate, pressure, and laser fluence. Enhancement of production is achieved by rastering the graphite target and by increasing the target surface temperature with a cw laser. In-situ diagnostics during production included time resolved passive emission and laser induced fluorescence from the plume. The improvement of the purity by a variety of steps in the purification process is monitored by characterization techniques including SEM, TEM, Raman, UV-VIS-NIR and TGA. A recently established NASA-JSC protocol for SWCNT characterization is undergoing revision with feedback from nanotube community. Efforts at JSC over the past five years in composites have centered on structural polymer/nanotube systems. Recent activities broadened this focus to multifunctional materials, supercapacitors, fuel cells, regenerable CO2 absorbers, electromagnetic shielding, radiation dosimetry and thermal management systems of interest for human space flight. Preliminary tests indicate improvement of performance in most of these applications because of the large Surface area as well as high electrical and thermal conductivity exhibited by SWCNTs. Comparison with existing technologies and possible future improvements in the SWCNT materials sill be presented.

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51.

PubMed

Messaoudi, Soumaya; Kergourlay, Gilles; Dalgalarrondo, Michèle; Choiset, Yvan; Ferchichi, Mounir; Prévost, Hervé; Pilet, Marie-France; Chobert, Jean-Marc; Manai, Mohamed; Dousset, Xavier

2012-10-01

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C(18) column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Production and physicochemical properties of recombinant Lactobacillus plantarum tannase.

PubMed

Curiel, José Antonio; Rodríguez, Héctor; Acebrón, Iván; Mancheño, José Miguel; De Las Rivas, Blanca; Muñoz, Rosario

2009-07-22

Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 degrees C. Addition of Ca(2+) to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

PubMed

Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst; Brøndsted, Lone

2017-01-01

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Purification of swine haptoglobin by affinity chromatography.

PubMed Central

Eurell, T E; Hall, W F; Bane, D P

1990-01-01

A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin. Images Fig. 2. Fig. 3. PMID:2123414

Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies

PubMed Central

Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G.V.T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.

2014-01-01

In this chapter, we concentrate on the production of high quality protein samples for NMR studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium, and outline our high-throughput strategies for producing high quality protein samples for nuclear magnetic resonance (NMR) studies. Our strategy is based on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6X-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (> 97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5,000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this paper describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research. PMID:21371586

Facile preparation of highly pure KF-ZrF4 molten salt

NASA Astrophysics Data System (ADS)

Zong, Guoqiang; Cui, Zhen-Hua; Zhang, Zhi-Bing; Zhang, Long; Xiao, Ji-Chang

2018-03-01

The preparation of highly pure KF-ZrF4 (FKZr) molten salt, a potential secondary coolant in molten salt reactors, was realized simply by heating a mixture of (NH4)2ZrF6 and KF. X-ray diffraction analysis indicated that the FKZr molten salt was mainly composed of KZrF5 and K2ZrF6. The melting point of the prepared FKZr molten salt was 420-422 °C under these conditions. The contents of all metal impurities were lower than 20 ppm, and the content of oxygen was lower than 400 ppm. This one-step protocol avoids the need for a tedious procedure to prepare ZrF4 and for an additional purification process to remove oxide impurities, and is therefore a convenient, efficient and economic preparation method for high-purity FKZr molten salt.

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

How valid are wearable physical activity trackers for measuring steps?

PubMed

An, Hyun-Sung; Jones, Gregory C; Kang, Seoung-Ki; Welk, Gregory J; Lee, Jung-Min

2017-04-01

Wearable activity trackers have become popular for tracking individual's daily physical activity, but little information is available to substantiate the validity of these devices in step counts. Thirty-five healthy individuals completed three conditions of activity tracker measurement: walking/jogging on a treadmill, walking over-ground on an indoor track, and a 24-hour free-living condition. Participants wore 10 activity trackers at the same time for both treadmill and over-ground protocol. Of these 10 activity trackers three were randomly given for 24-hour free-living condition. Correlations of steps measured to steps observed were r = 0.84 and r = 0.67 on a treadmill and over-ground protocol, respectively. The mean MAPE (mean absolute percentage error) score for all devices and speeds on a treadmill was 8.2% against manually counted steps. The MAPE value was higher for over-ground walking (9.9%) and even higher for the 24-hour free-living period (18.48%) on step counts. Equivalence testing for step count measurement resulted in a significant level within ±5% for the Fitbit Zip, Withings Pulse, and Jawbone UP24 and within ±10% for the Basis B1 band, Garmin VivoFit, and SenseWear Armband Mini. The results show that the Fitbit Zip and Withings Pulse provided the most accurate measures of step count under all three different conditions (i.e. treadmill, over-ground, and 24-hour condition), and considerable variability in accuracy across monitors and also by speeds and conditions.

Minimally complex ion traps as modules for quantum communication and computing

NASA Astrophysics Data System (ADS)

Nigmatullin, Ramil; Ballance, Christopher J.; de Beaudrap, Niel; Benjamin, Simon C.

2016-10-01

Optically linked ion traps are promising as components of network-based quantum technologies, including communication systems and modular computers. Experimental results achieved to date indicate that the fidelity of operations within each ion trap module will be far higher than the fidelity of operations involving the links; fortunately internal storage and processing can effectively upgrade the links through the process of purification. Here we perform the most detailed analysis to date on this purification task, using a protocol which is balanced to maximise fidelity while minimising the device complexity and the time cost of the process. Moreover we ‘compile down’ the quantum circuit to device-level operations including cooling and shuttling events. We find that a linear trap with only five ions (two of one species, three of another) can support our protocol while incorporating desirable features such as global control, i.e. laser control pulses need only target an entire zone rather than differentiating one ion from its neighbour. To evaluate the capabilities of such a module we consider its use both as a universal communications node for quantum key distribution, and as the basic repeating unit of a quantum computer. For the latter case we evaluate the threshold for fault tolerant quantum computing using the surface code, finding acceptable fidelities for the ‘raw’ entangling link as low as 83% (or under 75% if an additional ion is available).

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

DTIC Science & Technology

2016-08-01

Solubilization .................................................2  2.4  Denaturing Chromatography (Purification Step 1...Concentration Determination ................................................................4  2.10  Enzyme -Linked Immunosorbent Assay (ELISA...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in

Hyperentanglement purification using imperfect spatial entanglement.

PubMed

Wang, Tie-Jun; Mi, Si-Chen; Wang, Chuan

2017-02-06

As the interaction between the photons and the environment which will make the entangled photon pairs in less entangled states or even in mixed states, the security and the efficiency of quantum communication will decrease. We present an efficient hyperentanglement purification protocol that distills nonlocal high-fidelity hyper-entangled Bell states in both polarization and spatial-mode degrees of freedom from ensembles of two-photon system in mixed states using linear optics. Here, we consider the influence of the photon loss in the channel which generally is ignored in the conventional entanglement purification and hyperentanglement purification (HEP) schemes. Compared with previous HEP schemes, our HEP scheme decreases the requirement for nonlocal resources by employing high-dimensional mode-check measurement, and leads to a higher fidelity, especially in the range where the conventional HEP schemes become invalid but our scheme still can work.

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

PubMed

Lin, Chunbo; Shen, Maorong; Chen, Weiping; Li, Xiaofeng; Luo, Daoming; Cai, Jinhong; Yang, Yuan

2015-11-01

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Hormone Profiling in Plant Tissues.

PubMed

Müller, Maren; Munné-Bosch, Sergi

2017-01-01

Plant hormones are for a long time known to act as chemical messengers in the regulation of physiological processes during a plant's life cycle, from germination to senescence. Furthermore, plant hormones simultaneously coordinate physiological responses to biotic and abiotic stresses. To study the hormonal regulation of physiological processes, three main approaches have been used (1) exogenous application of hormones, (2) correlative studies through measurements of endogenous hormone levels, and (3) use of transgenic and/or mutant plants altered in hormone metabolism or signaling. A plant hormone profiling method is useful to unravel cross talk between hormones and help unravel the hormonal regulation of physiological processes in studies using any of the aforementioned approaches. However, hormone profiling is still particularly challenging due to their very low abundance in plant tissues. In this chapter, a sensitive, rapid, and accurate method to quantify all the five "classic" classes of plant hormones plus other plant growth regulators, such as jasmonates, salicylic acid, melatonin, and brassinosteroids is described. The method includes a fast and simple extraction procedure without time consuming steps as purification or derivatization, followed by optimized ultrahigh-performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS) analysis. This protocol facilitates the high-throughput analysis of hormone profiling and is applicable to different plant tissues.

Purification and Molecular Characterization of the Novel Highly Potent Bacteriocin TSU4 Produced by Lactobacillus animalis TSU4.

PubMed

Sahoo, Tapasa Kumar; Jena, Prasant Kumar; Patel, Amiya Kumar; Seshadri, Sriram

2015-09-01

Bacterial infections causing fish diseases and spoilage during fish food processing and storage are major concerns in aquaculture. Use of bacteriocins has recently been considered as an effective strategy for prevention of bacterial infections. A novel bacteriocin produced by Catla catla gut isolates, Lactobacillus animalis TSU4, designated as bacteriocin TSU4 was purified to homogeneity by a three-step protocol. The molecular mass of bacteriocin TSU4 was 4117 Da determined by Q-TOF LC/MS analysis. Its isoelectric point was ~9. Secondary conformation obtained by circular dichroism spectroscopy showed molecular conformation with significant proportions of the structure in α-helix (23.7 %) and β-sheets (17.1 %). N-terminal sequencing was carried out by the Edman degradation method; partial sequence identified was NH2-SMSGFSKPHD. Bacteriocin TSU4 exhibited a wide range of antimicrobial activity, pH and thermal stability. It showed a bacteriocidal mode of action against the indicator strain Aeromonas hydrophila MTCC 646. Bacteriocin TSU4 is the first reported bacteriocin produced by fish isolate Lactobacillus animalis. The characterization of bacteriocin TSU4 suggested that it is a novel bacteriocin with potential value against infections of bacteria such as A. hydrophila MTCC 646 and Pseudomonas aeruginosa MTCC 1688 and application to prevent spoilage during food preservation.

Template-Free Electroless Plating of Gold Nanowires: Direct Surface Functionalization with Shape-Selective Nanostructures for Electrochemical Applications.

PubMed

Muench, Falk; Schaefer, Sandra; Hagelüken, Lorenz; Molina-Luna, Leopoldo; Duerrschnabel, Michael; Kleebe, Hans-Joachim; Brötz, Joachim; Vaskevich, Alexander; Rubinstein, Israel; Ensinger, Wolfgang

2017-09-13

Metal nanowires (NWs) represent a prominent nanomaterial class, the interest in which is fueled by their tunable properties as well as their excellent performance in, for example, sensing, catalysis, and plasmonics. Synthetic approaches to obtain metal NWs mostly produce colloids or rely on templates. Integrating such nanowires into devices necessitates additional fabrication steps, such as template removal, nanostructure purification, or attachment. Here, we describe the development of a facile electroless plating protocol for the direct deposition of gold nanowire films, requiring neither templates nor complex instrumentation. The method is general, producing three-dimensional nanowire structures on substrates of varying shape and composition, with different seed types. The aqueous plating bath is prepared by ligand exchange and partial reduction of tetrachloroauric acid in the presence of 4-dimethylaminopyridine and formaldehyde. Gold deposition proceeds by nucleation of new grains on existing nanostructure tips and thus selectively produces curvy, polycrystalline nanowires of high aspect ratio. The nanofabrication potential of this method is demonstrated by producing a sensor electrode, whose performance is comparable to that of known nanostructures and discussed in terms of the catalyst architecture. Due to its flexibility and simplicity, shape-selective electroless plating is a promising new tool for functionalizing surfaces with anisotropic metal nanostructures.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

PubMed Central

Man-Kupisinska, Aleksandra; Michalski, Mateusz; Maciejewska, Anna; Swierzko, Anna S.; Cedzynski, Maciej; Lugowski, Czeslaw; Lukasiewicz, Jolanta

2016-01-01

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3. PMID:27232184

Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

PubMed Central

2014-01-01

Background Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Results Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. Conclusions This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS. PMID:24980254

Semiconductor grade, solar silicon purification project

NASA Technical Reports Server (NTRS)

Ingle, W. M.; Rosler, R. R.; Thompson, S. W.; Chaney, R. E.

1979-01-01

Experimental apparatus and procedures used in the development of a 3-step SiF2(x) polymer transport purification process are described. Both S.S.M.S. and E.S. analysis demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). Recent electrical analysis via crystal growth reveals that the product contains compensated phosphorus and boron. The low projected product cost and short energy payback time suggest that the economics of this process will result in a cost less than the goal of $10/Kg(1975 dollars). The process appears to be readily scalable to a major silicon purification facility.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process. PMID:21589929

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique.

PubMed

Snyman, Celia; Elliott, Edith

2011-12-15

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components. Copyright © 2011 Elsevier Inc. All rights reserved.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

PubMed Central

2014-01-01

Background There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes. PMID:24755191

Preparation of BAC libraries from marine microbial populations.

PubMed

Sabehi, Gazalah; Béjà, Oded

2013-01-01

A protocol is presented here for the construction of BAC (bacterial artificial chromosome) libraries from planktonic microbial communities collected in marine environments. The protocol describes the collection and preparation of the planktonic microbial cells, high molecular weight DNA purification from those cells, the preparation of the BAC vector, and the special ligation and electrotransformation procedures required for successful library preparation. With small modifications, this protocol can be applied to microbes collected from other environments. © 2013 Elsevier Inc. All rights reserved.

Water: A Recycling Success Story.

ERIC Educational Resources Information Center

Swinehart, Rebecca, Ed.

1995-01-01

This activity involves elementary students in simulating water purification techniques by finding ways to clear up soapy water. An introduction discusses water use and conservation. Materials needed and step-by-step procedure are provided. (LZ)

Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles.

PubMed

Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio

2015-01-28

Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

Preparative isolation, fast centrifugal partition chromatography purification and biological activity of cajaflavanone from Derris ferruginea stems.

PubMed

Morel, Sylvie; Landreau, Anne; Nguyen, Van Hung; Derbré, Séverine; Grellier, Philippe; Pape, Patrice Le; Pagniez, Fabrice; Litaudon, Marc; Richomme, Pascal

2012-01-01

The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. To develop an efficient preparative isolation procedure for bioactive cajaflavanone. Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC. Copyright © 2011 John Wiley & Sons, Ltd.

Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.

PubMed

Rouse, Sarah L; Hawthorne, Wlliam J; Lambert, Sebastian; Morgan, Marc L; Hare, Stephen A; Matthews, Stephen

2016-12-01

Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF 106-430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.

PREPARATIVE ISOLATION AND PURIFICATION OF THREE GLYCINE-CONJUGATED CHOLIC ACIDS FROM PULVIS FELLIS SUIS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY COUPLED WITH ELSD DETECTION.

PubMed

He, Jiao; Li, Jing; Sun, Wenji; Zhang, Tianyou; Ito, Yoichiro

2012-01-01

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was developed for preparative isolation and purification of three glycine-conjugated cholic acids, glycochenodeoxycholic acid (GCDCA), glycohyodeoxycholic acid (GHDCA) and glycohyocholic acid (GHCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The separation was performed with a two-phase solvent system consisted of chloroform-methanol-water-acetic acid (65:30:10:1.5, v/v/v/v) by eluting the lower phase in the head-to-tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 2 ml/min and 25 °C, respectively. In a single operation, 33 mg of GCDCA, 38 mg of GHDCA and 23 mg of GHCA were obtained from 200 mg of crude extract with the purity of 95.65%, 96.72% and 96.63%, respectively, in one step separation. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three glycine-conjugated cholic acids were identified by ESI-MS, (1)H NMR and (13)C NMR.

PREPARATIVE ISOLATION AND PURIFICATION OF THREE GLYCINE-CONJUGATED CHOLIC ACIDS FROM PULVIS FELLIS SUIS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY COUPLED WITH ELSD DETECTION

PubMed Central

He, Jiao; Li, Jing; Sun, Wenji; Zhang, Tianyou; Ito, Yoichiro

2011-01-01

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was developed for preparative isolation and purification of three glycine-conjugated cholic acids, glycochenodeoxycholic acid (GCDCA), glycohyodeoxycholic acid (GHDCA) and glycohyocholic acid (GHCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The separation was performed with a two-phase solvent system consisted of chloroform-methanol-water-acetic acid (65:30:10:1.5, v/v/v/v) by eluting the lower phase in the head-to-tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 2 ml/min and 25 °C, respectively. In a single operation, 33 mg of GCDCA, 38 mg of GHDCA and 23 mg of GHCA were obtained from 200 mg of crude extract with the purity of 95.65%, 96.72% and 96.63%, respectively, in one step separation. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three glycine-conjugated cholic acids were identified by ESI-MS, 1H NMR and 13C NMR. PMID:23008527

Parallel production and verification of protein products using a novel high-throughput screening method.

PubMed

Tegel, Hanna; Yderland, Louise; Boström, Tove; Eriksson, Cecilia; Ukkonen, Kaisa; Vasala, Antti; Neubauer, Peter; Ottosson, Jenny; Hober, Sophia

2011-08-01

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.

PubMed

Demir, Hülya; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2003-02-01

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Large-Scale Expansion of Human iPSC-Derived Skeletal Muscle Cells for Disease Modeling and Cell-Based Therapeutic Strategies.

PubMed

van der Wal, Erik; Herrero-Hernandez, Pablo; Wan, Raymond; Broeders, Mike; In 't Groen, Stijn L M; van Gestel, Tom J M; van IJcken, Wilfred F J; Cheung, Tom H; van der Ploeg, Ans T; Schaaf, Gerben J; Pijnappel, W W M Pim

2018-06-05

Although skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 10 11 -fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of acid α-glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/Cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

PubMed

Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

2017-04-01

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, β-lactoglobulin.

PubMed

Ritala, A; Leelavathi, S; Oksman-Caldentey, K-M; Reddy, V S; Laukkanen, M-L

2014-06-01

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for β-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.

Purification of α-Synuclein from Human Brain Reveals an Instability of Endogenous Multimers as the Protein Approaches Purity

PubMed Central

2015-01-01

Despite two decades of research, the structure–function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson’s disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification. PMID:25490121

Multi-copy entanglement purification with practical spontaneous parametric down conversion sources

NASA Astrophysics Data System (ADS)

Zhang, Shuai-Shuai; Shu, Qi; Zhou, Lan; Sheng, Yu-Bo

2017-06-01

Entanglement purification is to distill the high quality entanglement from the low quality entanglement with local operations and classical communications. It is one of the key technologies in long-distance quantum communication. We discuss an entanglement purification protocol (EPP) with spontaneous parametric down conversion (SPDC) sources, in contrast to previous EPP with multi-copy mixed states, which requires ideal entanglement sources. We show that the SPDC source is not an obstacle for purification, but can benefit the fidelity of the purified mixed state. This EPP works for linear optics and is feasible in current experiment technology. Project supported by the National Natural Science Foundation of China (Grant Nos. 11474168 and 61401222), the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20151502), the Qing Lan Project in Jiangsu Province, China, and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

Characterization of synthetic macroporous ion-exchange resins in low-pressure cartridges and columns. Evaluation of the performance of Macro-Prep 50 S resin in the purification of anti-Klenow antibodies from goat serum.

PubMed

Dunn, L; Abouelezz, M; Cummings, L; Navvab, M; Ordunez, C; Siebert, C J; Talmadge, K W

1991-07-12

Three ion-exchange materials and one hydrophobic-interaction chromatography packing, based on a rigid macroporous polymer with large, relatively uniform pores, have been evaluated for low-pressure liquid chromatography of antibodies. These sorbents have high capacities for both small and large proteins and are mechanically, chemically, and thermally stable. Macro-Prep 50 S. CM and Q ion-exchange materials are strongly acidic, weakly acidic, and strongly basic, respectively. Protein binding and recovery, pressure-flow properties, and chemical and thermal stability were determined for each sorbent. A rapid, two-step method for the purification of anti-Klenow antibodies from goat serum was developed, based on the Macro-Prep 50 S strong-acid cation-exchange material and the Econo-Pac HIC prepacked hydrophobic-interaction cartridge.

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data.

PubMed

Eddhif, Balkis; Guignard, Nadia; Batonneau, Yann; Clarhaut, Jonathan; Papot, Sébastien; Geffroy-Rodier, Claude; Poinot, Pauline

2018-04-01

The data presented here are related to the research paper entitled "Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses" (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods.

PubMed

Wunschel, David S; Melville, Angela M; Ehrhardt, Christopher J; Colburn, Heather A; Victry, Kristin D; Antolick, Kathryn C; Wahl, Jon H; Wahl, Karen L

2012-05-07

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

Comparison of Diafiltration and Size-Exclusion Chromatography to Recover Hemicelluloses From Process Water From Thermomechanical Pulping of Spruce

NASA Astrophysics Data System (ADS)

Andersson, Alexandra; Persson, Tobias; Zacchi, Guido; Stålbrand, Henrik; Jönsson, Ann-Sofi

Hemicelluloses constitute one of the most abundant renewable resources on earth. To increase their utilization, the isolation of hemicelluloses from industrial biomass side-streams would be beneficial. A method was investigated to isolate hemicelluloses from process water from a thermomechanical pulp mill. The method consists of three steps: removal of solids by microfiltration, preconcentration of the hemicelluloses by ultrafiltration, and purification by either size-exclusion chromatography (SEC) or diafiltration. The purpose of the final purification step is to separate hemicelluloses from small oligosaccharides, monosaccharides, and salts. The ratio between galactose, glucose, and mannose in oligo- and polysaccharides after preconcentration was 0.8∶1∶2.8, which is similar to that found in galactoglucomannan. Continuous diafiltration was performed using a composite fluoro polymer membrane with cutoff of 1000 Da. After diafiltration with four diavolumes the purity of the hemicelluloses was 77% (gram oligo- and polysaccharides/ gram total dissolved solids) and the recovery was 87%. Purification by SEC was performed with 5, 20, and 40% sample loadings, respectively and a flow rate of 12 or 25 mL/min (9 or 19 cm/h). The purity of hemicelluloses after SEC was approx 82%, and the recovery was above 99%. The optimal sample load and flow rate were 20% and 25 mL/min, respectively. The process water from thermomechanical pulping of spruce is inexpensive. Thus, the recovery of hemicelluloses is not of main importance. If the purity of 77%, obtained with diafiltration, is sufficient for the utilization of the hemicelluloses, diafiltration probably offers a less expensive alternative in this application.

Purification of Arp2/3 complex from Saccharomyces cerevisiae

PubMed Central

Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

2014-01-01

Summary Much of cellular control over actin dynamics comes through regulation of actin filament initiation. At the molecular level, this is accomplished through a collection of cellular protein machines, called actin nucleation factors, which position actin monomers to initiate a new actin filament. The Arp2/3 complex is a principal actin nucleation factor used throughout the eukaryotic family tree. The budding yeast Saccharomyces cerevisiae has proven to be not only an excellent genetic platform for the study of the Arp2/3 complex, but also an excellent source for the purification of endogenous Arp2/3 complex. Here we describe a protocol for the preparation of endogenous Arp2/3 complex from wild type Saccharomyces cerevisiae. This protocol produces material suitable for biochemical study, and yields milligram quantities of purified Arp2/3 complex. PMID:23868593

Purification of Bacteriophages Using Anion-Exchange Chromatography.

PubMed

Vandenheuvel, Dieter; Rombouts, Sofie; Adriaenssens, Evelien M

2018-01-01

In bacteriophage research and therapy, most applications ask for highly purified phage suspensions. The standard technique for this is ultracentrifugation using cesium chloride gradients. This technique is cumbersome, elaborate and expensive. Moreover, it is unsuitable for the purification of large quantities of phage suspensions.The protocol described here, uses anion-exchange chromatography to bind phages to a stationary phase. This is done using an FLPC system, combined with Convective Interaction Media (CIM ® ) monoliths. Afterward, the column is washed to remove impurities from the CIM ® disk. By using a buffer solution with a high ionic strength, the phages are subsequently eluted from the column and collected. In this way phages can be efficiently purified and concentrated.This protocol can be used to determine the optimal buffers, stationary phase chemistry and elution conditions, as well as the maximal capacity and recovery of the columns.

Ultrasound assisted three phase partitioning of a fibrinolytic enzyme.

PubMed

Avhad, Devchand N; Niphadkar, Sonali S; Rathod, Virendra K

2014-03-01

The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55-70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique. Copyright © 2013 Elsevier B.V. All rights reserved.

The '333' integrated strategy for effective pollution control and its application to the heavily polluted Jialu River in north China.

PubMed

Huang, Yu; Sun, Jie; Li, Aimin; Xie, Xianchuan

2018-05-01

In this study, an integrated approach named the '333' strategy was applied to pollution control in the Jialu River, in northern China, which is heavily burdened with anthropogenic pollution. Due to a deficiency of the natural ecological inflow, the Jialu River receives predominantly industrial and municipal effluent. The '333' strategy is composed of three steps of pollution control including industrial point-source pollution control, advanced treatment of municipal wastewater, and ecological restoration; three increased stringency emission standards; and three stages of reclamation. Phase 1 of the '333' strategy focuses on industrial point-source pollution control; phase 2 aims to harness municipal wastewater and minimize sewage effluents using novel techniques for advanced water purification; phase 3 of the '333' strategy focuses on the further purification of effluents flowing into Jialu River with the employment of an engineering-based ecological restoration project. The application of the '333' strategy resulted in the development of novel techniques for water purification including modified magnetic resins (NDMP resin), a two-stage internal circulation anaerobic reactor (IC reactor) and an ecological restoration system. The results indicate that water quality in the river was significantly improved, with increased concentrations of dissolved oxygen (DO), as well as reduction of COD by 42.8% and NH 3 -N by 61.4%. In addition, it was observed that the total population of phytoplankton in treated river water notably increased from only one prior to restoration to 8 following restoration. This system also provides a tool for pollution control of other similar industrial and anthropogenic source polluted rivers.

Efficient entanglement distillation without quantum memory.

PubMed

Abdelkhalek, Daniela; Syllwasschy, Mareike; Cerf, Nicolas J; Fiurášek, Jaromír; Schnabel, Roman

2016-05-31

Entanglement distribution between distant parties is an essential component to most quantum communication protocols. Unfortunately, decoherence effects such as phase noise in optical fibres are known to demolish entanglement. Iterative (multistep) entanglement distillation protocols have long been proposed to overcome decoherence, but their probabilistic nature makes them inefficient since the success probability decays exponentially with the number of steps. Quantum memories have been contemplated to make entanglement distillation practical, but suitable quantum memories are not realised to date. Here, we present the theory for an efficient iterative entanglement distillation protocol without quantum memories and provide a proof-of-principle experimental demonstration. The scheme is applied to phase-diffused two-mode-squeezed states and proven to distil entanglement for up to three iteration steps. The data are indistinguishable from those that an efficient scheme using quantum memories would produce. Since our protocol includes the final measurement it is particularly promising for enhancing continuous-variable quantum key distribution.

Efficient entanglement distillation without quantum memory

PubMed Central

Abdelkhalek, Daniela; Syllwasschy, Mareike; Cerf, Nicolas J.; Fiurášek, Jaromír; Schnabel, Roman

2016-01-01

Entanglement distribution between distant parties is an essential component to most quantum communication protocols. Unfortunately, decoherence effects such as phase noise in optical fibres are known to demolish entanglement. Iterative (multistep) entanglement distillation protocols have long been proposed to overcome decoherence, but their probabilistic nature makes them inefficient since the success probability decays exponentially with the number of steps. Quantum memories have been contemplated to make entanglement distillation practical, but suitable quantum memories are not realised to date. Here, we present the theory for an efficient iterative entanglement distillation protocol without quantum memories and provide a proof-of-principle experimental demonstration. The scheme is applied to phase-diffused two-mode-squeezed states and proven to distil entanglement for up to three iteration steps. The data are indistinguishable from those that an efficient scheme using quantum memories would produce. Since our protocol includes the final measurement it is particularly promising for enhancing continuous-variable quantum key distribution. PMID:27241946

Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey.

PubMed

Genc, Berna; Nadaroglu, Hayrunnisa; Adiguzel, Ahmet; Baltaci, Ozkan

2015-11-01

In the present study, cellulase was purified and characterized from Anoxybacillus gonensis (Gen bank Number: KM596794) which was isolated and characterized from Agri Diyadin Hot spring. It was found to synthesize cellulase which had a wide range of industrial applications. Twenty four-hour-cultured bacteria induced cellulase production and specific activities during the purification steps were 1.47, 81.06 and 109.4 EU mg(-1) protein at crude extract, ammonium sulphate precipitated and DEAE-Sephadex purification steps. The highest enzyme activity was observed at 50°C and the optimum range of pH was 3-10. Molecular weight of enzyme was determined approximately 40kDa. The kinetic parameters of cellulase against carboxymethylcellulose (CMC) were 153.4 pmol min(-1) mg for Vmax and 0.46mM for Km. Among effectors of the enzyme, Zn2+, Ca2+, Co2+ and EDTA decreased enzyme activity.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

PubMed

Carvalho, Rimenys J; Cruz, Thayana A

2018-01-01

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

Plasma fibronectin: three steps to purification and stability.

PubMed

Poulouin, L; Gallet, O; Rouahi, M; Imhoff, J M

1999-10-01

Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis. Copyright 1999 Academic Press.

Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

NASA Astrophysics Data System (ADS)

Zimny, Philip; Juncker, David; Reisner, Walter

Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

Purification of NAD glycohydrolase from Agkistrodon acutus venom.

PubMed

Wu, Shuang Ding; Liu, Yanli; Xu, Xiaolong; Zhu, Zhengang

2002-07-01

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.

Recovery and purification process development for monoclonal antibody production

PubMed Central

Ma, Junfen; Winter, Charles; Bayer, Robert

2010-01-01

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

PubMed

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Entanglement concentration and purification of two-mode squeezed microwave photons in circuit QED

NASA Astrophysics Data System (ADS)

Zhang, Hao; Alsaedi, Ahmed; Hayat, Tasawar; Deng, Fu-Guo

2018-04-01

We present a theoretical proposal for a physical implementation of entanglement concentration and purification protocols for two-mode squeezed microwave photons in circuit quantum electrodynamics (QED). First, we give the description of the cross-Kerr effect induced between two resonators in circuit QED. Then we use the cross-Kerr media to design the effective quantum nondemolition (QND) measurement on microwave-photon number. By using the QND measurement, the parties in quantum communication can accomplish the entanglement concentration and purification of nonlocal two-mode squeezed microwave photons. We discuss the feasibility of our schemes by giving the detailed parameters which can be realized with current experimental technology. Our work can improve some practical applications in continuous-variable microwave-based quantum information processing.

Fast Entanglement Establishment via Local Dynamics for Quantum Repeater Networks

NASA Astrophysics Data System (ADS)

Gyongyosi, Laszlo; Imre, Sandor

Quantum entanglement is a necessity for future quantum communication networks, quantum internet, and long-distance quantum key distribution. The current approaches of entanglement distribution require high-delay entanglement transmission, entanglement swapping to extend the range of entanglement, high-cost entanglement purification, and long-lived quantum memories. We introduce a fundamental protocol for establishing entanglement in quantum communication networks. The proposed scheme does not require entanglement transmission between the nodes, high-cost entanglement swapping, entanglement purification, or long-lived quantum memories. The protocol reliably establishes a maximally entangled system between the remote nodes via dynamics generated by local Hamiltonians. The method eliminates the main drawbacks of current schemes allowing fast entanglement establishment with a minimized delay. Our solution provides a fundamental method for future long-distance quantum key distribution, quantum repeater networks, quantum internet, and quantum-networking protocols. This work was partially supported by the GOP-1.1.1-11-2012-0092 project sponsored by the EU and European Structural Fund, by the Hungarian Scientific Research Fund - OTKA K-112125, and by the COST Action MP1006.

The development of a new three-step protocol to determine the efficacy of disinfectant wipes on surfaces contaminated with Staphylococcus aureus.

PubMed

Williams, G J; Denyer, S P; Hosein, I K; Hill, D W; Maillard, J-Y

2007-12-01

We developed a three-step protocol to quantify the efficacy of disinfectant wipes, their ability to remove and prevent microbial transfer from surfaces and their overall antimicrobial activity. Meticillin-resistant (MRSA) or -susceptible (MSSA) Staphylococcus aureus (6-7 log(10)cfu) were inoculated onto stainless steel discs with or without organic load and dried. Grapefruit extract-containing test wipes and unmedicated control wipes were used. In step 1, wipes were mechanically rotated against surfaces for 10s at 60rpm, exerting a weight of 100+/-5g. Bacterial removal was assessed by transferring the steel discs to neutraliser, resuspending and counting remaining bacteria. In step 2, bacterial transfer from wipes was assessed by eight consecutive mechanical adpression transfers to agar/neutraliser plates. Step 3 was the measurement of antimicrobial activity by direct inoculation of the wipes for 10s followed by neutralisation and enumeration. Test wipes achieved a significantly higher bacterial cell removal than control wipes on all surfaces (P<0.05). The low bactericidal activity of the wipes (<1 log(10) reduction when directly inoculated) and the subsequent survival of bacteria on the wipes, however, led to repeated microbial transfer when initially high contamination levels were present. There were no differences between MRSA and MSSA in removal, transfer or antimicrobial activity. The three-step method is a useful tool for developing future guidelines to assess the ability of wipes to disinfect surfaces.

Purification of FLAG-tagged Secreted Proteins from Mammalian Cells

PubMed Central

Itakura, Eisuke; Chen, Changchun; de Bono, Mario

2017-01-01

This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays. PMID:29075655

An improved procedure for separation/purification of boron from complex matrices and high-precision measurement of boron isotopes by positive thermal ionization and multicollector inductively coupled plasma mass spectrometry.

PubMed

Wei, Hai-Zhen; Jiang, Shao-Yong; Hemming, N Gary; Yang, Jing-Hong; Yang, Tao; Wu, He-Pin; Yang, Tang-Li; Yan, Xiong; Pu, Wei

2014-06-01

In order to eliminate boron loss and potential isotopic fractionation during chemical pretreatment of natural samples with complex matrices, a three-column ion-exchange separation/purification procedure has been modified, which ensures more than 98% recovery of boron from each step for a wide range of sample matrices, and is applicable for boron isotope analysis by both TIMS and MC-ICP-MS. The PTIMS-Cs2BO2(+)-static double collection method was developed, ensuring simultaneous collection of (133)Cs2(11)B(16)O2(+)(m/z 309) and (133)Cs2(10)B(16)O2(+) (m/z 308) ions in adjacent H3-H4 Faraday cups with typical zoom optics parameters (Focus Quad: 15 V, Dispersion Quad: -85 V). The external reproducibilities of the measured (11)B/(10)B ratios of the NIST 951 boron standard solutions of 1000 ng, 100 ng and 10 ng of boron by PTIMS method are ±0.06‰, ±0.16‰ and ±0.25‰, respectively, which indicates excellent precision can be achieved for boron isotope measurement at nanogram level boron in natural samples. An on-peak zero blank correction procedure was employed to correct the residual boron signals effect in MC-ICP-MS, which gives consistent δ(11)B values with a mean of 39.66±0.35‰ for seawater in the whole range of boron content from 5 ppb to 200 ppb, ensuring accurate boron isotope analysis in few ppb boron. With the improved protocol, consistent results between TIMS and MC-ICP-MS data were obtained in typical geological materials within a wide span of δ(11)B values ranging from -25‰ to +40‰. Copyright © 2014 Elsevier B.V. All rights reserved.

Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures.

PubMed

Koussa, Mounir A; Sotomayor, Marcos; Wong, Wesley P

2014-05-15

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. Copyright © 2014. Published by Elsevier Inc.

Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures

PubMed Central

Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.

2014-01-01

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. PMID:24568941

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

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

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus,more » led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.« less

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching*

PubMed Central

Billen, Bert; Brams, Marijke; Debaveye, Sarah; Remeeva, Alina; Alpizar, Yeranddy A.; Waelkens, Etienne; Kreir, Mohamed; Brüggemann, Andrea; Talavera, Karel; Nilius, Bernd; Voets, Thomas; Ulens, Chris

2015-01-01

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. PMID:25829496

Amplification of Mycoplasma haemofelis DNA by a PCR for point-of-care use.

PubMed

Hawley, Jennifer; Yaaran, Tal; Maurice, Sarah; Lappin, Michael R

2018-01-01

We compared a qualitative in-clinic (IC)-PCR for the detection of Mycoplasma haemofelis DNA with the results of a commercial qualitative laboratory-based, conventional (c)PCR. In order to determine the specificity of both tests, Bartonella spp. samples were included. Forty-three previously tested blood samples with known PCR results for hemoplasmas and Bartonella spp. were selected. The samples were split between 2 laboratories. At the first laboratory, DNA was purified and run on 2 cPCR assays for the detection of hemoplasmas and Bartonella spp. At the second laboratory, DNA was purified using 2 purification protocols and both run in the IC-PCR assay. The cPCR results confirmed that 18 samples were positive for M. haemofelis, 5 for ' Candidatus M. haemominutum', 8 for Bartonella henselae, 2 for Bartonella clarridgeiae, and 10 were negative for both genera. No mixed infections were observed. The IC-PCR assay for the detection of M. haemofelis had a sensitivity of 94.4% and specificity of 96%, when using the same DNA purification method as the first laboratory. Using the second purification method, the sensitivity of the IC-PCR assay was 77.8% and specificity was 96%. Bartonella species were not detected by the IC-PCR M. haemofelis assay. The IC-PCR assay decreased the amount of time to final result compared to a cPCR assay.

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Laboratory information management system for membrane protein structure initiative--from gene to crystal.

PubMed

Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

2008-12-01

Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

Biolistic Transformation of Wheat.

PubMed

Tassy, Caroline; Barret, Pierre

2017-01-01

The wheat genome encodes some 100,000 genes. To understand how the expression of these genes is regulated it will be necessary to carry out many genetic transformation experiments. Robust protocols that allow scientists to transform a wide range of wheat genotypes are therefore required. In this chapter, we describe a protocol for biolistic transformation of wheat that uses immature embryos and small quantities of DNA cassettes. An original method for DNA cassette purification is also described. This protocol can be used to transform a wide range of wheat genotypes and other related species.

Modular synthesis of thiazoline and thiazole derivatives by using a cascade protocol.

PubMed

Alsharif, Zakeyah A; Alam, Mohammad A

2017-01-01

Thiazolines and thiazoles are an integral part of numerous natural products, a number of drugs, and many useful molecules such as ligands for metal catalysis. We report the first common synthetic protocol for the synthesis of thiazoles and thiazolines. Novel molecules are efficiently synthesized by using readily available and inexpensive substrates. The reaction conditions are mild and pure products are obtained without work-up and column purification.

Fully automated SPE-based synthesis and purification of 2-[18F]fluoroethyl-choline for human use.

PubMed

Schmaljohann, Jörn; Schirrmacher, Esther; Wängler, Björn; Wängler, Carmen; Schirrmacher, Ralf; Guhlke, Stefan

2011-02-01

2-[(18)F]Fluoroethyl-choline ([(18)F]FECH) is a promising tracer for the detection of prostate cancer as well as brain tumors with positron emission tomography (PET). [(18)F]FECH is actively transported into mammalian cells, becomes phosphorylated by choline kinase and gets incorporated into the cell membrane after being metabolized to phosphatidylcholine. So far, its synthesis is a two-step procedure involving at least one HPLC purification step. To allow a wider dissemination of this tracer, finding a purification method avoiding HPLC is highly desirable and would result in easier accessibility and more reliable production of [(18)F]FECH. [(18)F]FECH was synthesized by reaction of 2-bromo-1-[(18)F]fluoroethane ([(18)F]BFE) with dimethylaminoethanol (DMAE) in DMSO. We applied a novel and very reliable work-up procedure for the synthesis of [(18)F]BFE. Based on a combination of three different solid-phase cartridges, the purification of [(18)F]BFE from its precursor 2-bromoethyl-4-nitrobenzenesulfonate (BENos) could be achieved without using HPLC. Following the subsequent reaction of the purified [(18)F]BFE with DMAE, the final product [(18)F]FECH was obtained as a sterile solution by passing the crude reaction mixture through a combination of two CM plus cartridges and a sterile filter. The fully automated synthesis was performed using as well a Raytest SynChrom module (Raytest, Germany) or a Scintomics HotboxIII module (Scintomics, Germany). The radiotracer [(18)F]FECH can be synthesized in reliable radiochemical yields (RCY) of 37±5% (Synchrom module) and 33±5% (Hotbox III unit) in less than 1 h using these two fully automated commercially available synthesis units without HPLC involvement for purification. Detailed quality control of the final injectable [(18)F]FECH solution proved the high radiochemical purity and the absence of Kryptofix2.2.2, DMAE and DMSO used in the course of synthesis. Sterility and bacterial endotoxin testing following standard procedures verified that the described production method for [(18)F]FECH is suitable for human applications. The routine production of [(18)F]FECH with sufficient RCYs was established by reliable and fast solid-phase extraction purifications of both the secondary labeling precursor [(18)F]BFE and the final product [(18)F]FECH, avoiding complex and sensitive HPLC equipment. The purity of the product was >95%, rendering the tracer suitable for human application. The newly developed purification procedure for [(18)F]BFE significantly reduces the complexity of the automated synthesis unit, hence reducing the cost for routine production in a clinical setup and allowing easy transfer to different synthesis modules. Copyright © 2011 Elsevier Inc. All rights reserved.

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

PubMed

de Moura, Andréa A; Kayano, Anderson M; Oliveira, George A; Setúbal, Sulamita S; Ribeiro, João G; Barros, Neuza B; Nicolete, Roberto; Moura, Laura A; Fuly, Andre L; Nomizo, Auro; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M; Calderon, Leonardo A

2014-01-01

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Preparative isolation and purification of capsaicin and dihydrocapsaicin from Capsici Fructus using supercritical fluid extraction combined with high speed countercurrent chromatography.

PubMed

Yan, Rongwei; Zhao, Leilei; Tao, Junfei; Zou, Yong; Xu, Xinjun

2018-05-01

Supercritical fluid extraction with CO 2 (SFE-CO 2 ) was utilized for extraction of capsaicin (CA) and dihydrocapsaicin (DHCA) from Capsici Fructus, and then a two-step enrichment method for separating capsaicinoids from SFE-CO 2 extracts was developed. The process involved extraction with aqueous methanol and crystallization by alkali extraction and acid precipitation. Finally, a consecutive high-speed countercurrent chromatography (HSCCC) separation method was successfully applied in the purification of CA and DHCA from capsaicinoid crystal. The extraction pressure, extraction temperature and volume of co-solvent were optimized at 33 MPa, 41 °C and 75 mL, respectively, using response surface methodology; the extraction rates of CA and DHCA were about 93.18% and 93.49%, respectively. 407.43 mg capsaicinoid crystal was isolated from the SFE-CO 2 extracts obtained from 100 g capsicum powder by the two-step enrichment method. About 506 mg and 184 mg CA and DHCA with purities up to 98.31% and 96.68%, respectively, were obtained from 1 g capsaicinoid crystal in one HSCCC of three consecutive sample loadings without exchanging any solvent system. This method comprising SFE-CO 2 , a two-step enrichment and HSCCC was efficient, powerful and practical for the large-scale preparation of CA and DHCA from Capsici Fructus with high purity and high yield. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

PubMed

Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

2016-02-01

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cyanogenesis in Cassava1

PubMed Central

White, Wanda L.B.; Arias-Garzon, Diana I.; McMahon, Jennifer M.; Sayre, Richard T.

1998-01-01

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products. PMID:9536038

Design and analysis of siloxanes removal by adsorption from landfill gas for waste-to-energy processes.

PubMed

Elwell, Anthony C; Elsayed, Nada H; Kuhn, John N; Joseph, Babu

2018-03-01

Separation of volatile methyl siloxanes from landfill gas using fixed adsorption beds was modeled with the objective of identifying appropriate technology and the economics associated with this purification step. A general adsorption model assuming plug flow and radial symmetry was developed and used to conduct a parametric sweep of 162 unique cases. The varied parameters were adsorbent type (activated carbon and silica gel), bed height (3.05-9.15 m/10-30 ft), inlet siloxane concentration (5-15 mg/m 3 ), moisture content (0-100% relative humidity at STP or RH), and siloxane tolerance limit (0.094-9.4 mg/m 3 ) that correlated to three distinct energy conversion technologies (electricity production using engines or fuels cells or catalytic conversion to liquid hydrocarbon fuels). Due to the detrimental effect of RH on siloxane absorption, the maximum allowable moisture content of LFG before purification is 50% RH and moisture removal processes are also required. The design calculations using a selected case study show that the adsorption bed height required needed for 6 months minimum breakthrough time for catalytic fuel production is twice that for engine applications. Fuel cell applications require 3 times the bed height compared to engine applications. However, the purification costs amounted to 94%, 16% and 52% of recovered product value for engine, liquefaction, and fuel cell applications, respectively indicating the need for a high value product to justify purification costs. The approaches and conclusions can be extended to specific process conditions for landfill gas purification and to other processes that use biogas produced from waste as a feedstock. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

PubMed

Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2002-05-01

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

PubMed Central

Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

2014-01-01

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

Overview of the purification of recombinant proteins.

PubMed

Wingfield, Paul T

2015-04-01

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

Three-input majority function as the unique optimal function for the bias amplification using nonlocal boxes

NASA Astrophysics Data System (ADS)

Mori, Ryuhei

2016-11-01

Brassard et al. [Phys. Rev. Lett. 96, 250401 (2006), 10.1103/PhysRevLett.96.250401] showed that shared nonlocal boxes with a CHSH (Clauser, Horne, Shimony, and Holt) probability greater than 3/+√{6 } 6 yield trivial communication complexity. There still exists a gap with the maximum CHSH probability 2/+√{2 } 4 achievable by quantum mechanics. It is an interesting open question to determine the exact threshold for the trivial communication complexity. Brassard et al.'s idea is based on recursive bias amplification by the three-input majority function. It was not obvious if another choice of function exhibits stronger bias amplification. We show that the three-input majority function is the unique optimal function, so that one cannot improve the threshold 3/+√{6 } 6 by Brassard et al.'s bias amplification. In this work, protocols for computing the function used for the bias amplification are restricted to be nonadaptive protocols or a particular adaptive protocol inspired by Pawłowski et al.'s protocol for information causality [Nature (London) 461, 1101 (2009), 10.1038/nature08400]. We first show an adaptive protocol inspired by Pawłowski et al.'s protocol, and then show that the adaptive protocol improves upon nonadaptive protocols. Finally, we show that the three-input majority function is the unique optimal function for the bias amplification if we apply the adaptive protocol to each step of the bias amplification.

The influence of polymer purification on photovoltaic device performance of a series of indacenodithiophene donor polymers.

PubMed

Ashraf, Raja Shahid; Schroeder, Bob C; Bronstein, Hugo A; Huang, Zhenggang; Thomas, Stuart; Kline, R Joseph; Brabec, Christoph J; Rannou, Patrice; Anthopoulos, Thomas D; Durrant, James R; McCulloch, Iain

2013-04-11

A series of low bandgap indacenodithiophene polymers is purified by recycling SEC in order to isolate narrow polydispersity fractions. This additional purification step is found to have a significant beneficial influence on the solar cell performance and the reasons for this performance increase are investigated. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Legionella pneumophila Toxin, Isolation and Purification

DTIC Science & Technology

1981-01-01

which dis- plays an in vivo lethality. The purification procedures involve acid precipitation, gel chromatography, and preparative isotachophoresis. The...Chymotrypsinogen A, Ribonuclease A, and Apoprotinin as markers. Preparation of antiserum One milliqram amounts of protein from Le jonella acid ...RESULTS Toxin isolation Step 1: Acid precipitation of crude toxin. 1.0 N HCl acid was slowly added to rapidly stirred crude toxin until pH 3.5 was

The Isolation of Invertase from Baker's Yeast: A Four-Part Exercise in Protein Purification and Characterization

ERIC Educational Resources Information Center

Timerman, Anthony P.; Fenrick, Angela M.; Zamis, Thomas M.

2009-01-01

A sequence of exercises for the isolation and characterization of invertase (E.C. 3.1.2.26) from baker's yeast obtained from a local grocery store is outlined. Because the enzyme is colorless, the use of colored markers and the sequence of purification steps are designed to "visualize" the process by which a colorless protein is selectively…

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project.

PubMed

de Hoogt, Ronald; Estrada, Marta F; Vidic, Suzana; Davies, Emma J; Osswald, Annika; Barbier, Michael; Santo, Vítor E; Gjerde, Kjersti; van Zoggel, Hanneke J A A; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M; Verschuren, Emmy W; Hickman, John; Graeser, Ralph

2017-11-21

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

PubMed Central

de Hoogt, Ronald; Estrada, Marta F.; Vidic, Suzana; Davies, Emma J.; Osswald, Annika; Barbier, Michael; Santo, Vítor E.; Gjerde, Kjersti; van Zoggel, Hanneke J. A. A.; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M.; Verschuren, Emmy W.; Hickman, John; Graeser, Ralph

2017-01-01

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented. PMID:29160867

A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA.

PubMed

Vijayalakshmy, K; Kumar, P; Virmani, M; Pawaria, S; Lalaji, N S; Sharma, P; Rajendran, R; Yadav, P S; Kumar, D

2018-05-14

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP-coated silica colloidal solution (PVP-Si), silane-coated silica colloidal solution (Silane-Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane-Si and iodixanol compared to PVA-Si method. The combined guanidinium thiocyanate-phenol-chloroform (GTPC) with silica matrix (SM)-based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360-450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4-5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27-45 times. In summary, Silane-Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high-throughput analyses of buffalo sperm RNA. © 2018 Blackwell Verlag GmbH.

One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

PubMed

Caldinelli, Laura; Albani, Diego; Pollegioni, Loredano

2013-04-04

Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets

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

Cherezov, Vadim; Abola, Enrique; Stevens, Raymond C.

2015-11-30

G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extra-cellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success ofmore » using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here they describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data. The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.« less

Protein complex purification from Thermoplasma acidophilum using a phage display library.

PubMed

Hubert, Agnes; Mitani, Yasuo; Tamura, Tomohiro; Boicu, Marius; Nagy, István

2014-03-01

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. Copyright © 2013 Elsevier B.V. All rights reserved.

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

PubMed

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

PubMed

Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

2013-12-01

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

Purification of Tronoh Silica Sand via preliminary process of mechanical milling

NASA Astrophysics Data System (ADS)

H, Nazratulhuda; M, Othman

2016-02-01

The purification of Tronoh silica sand is an important step in expanding technical applications of this silica sand. However no research on purifying of Tronoh silica sand has been reported. This study is focused on ball milling technique as a preliminary technique for Tronoh silica sand purification. The objectives are to study the effect of ball milling to the purification of the silica sand and to analyze its characteristics after the ball milling process. The samples before and after milling process were analyzed by using XRF, XRD, SEM and TEM. Results showed that the purity of SiO2 was increased, the size of the particles has been reduced and the surface area has increased. The crystalline phases for the silica before and after 4 hour milling time were remained constant.

Adolescent pedometer protocols: examining reactivity, tampering and participants' perceptions.

PubMed

Scott, Joseph John; Morgan, Philip James; Plotnikoff, Ronald Cyril; Trost, Stewart Graeme; Lubans, David Revalds

2014-01-01

The aim of this study was to investigate adolescents' potential reactivity and tampering while wearing pedometers by comparing different monitoring protocols to accelerometer output. The sample included adolescents (N = 123, age range = 14-15 years) from three secondary schools in New South Wales, Australia. Schools were randomised to one of the three pedometer monitoring protocols: (i) daily sealed (DS) pedometer group, (ii) unsealed (US) pedometer group or (iii) weekly sealed (WS) pedometer group. Participants wore pedometers (Yamax Digi-Walker CW700, Yamax Corporation, Kumamoto City, Japan) and accelerometers (Actigraph GT3X+, Pensacola, USA) simultaneously for seven days. Repeated measures analysis of variance was used to examine potential reactivity. Bivariate correlations between step counts and accelerometer output were calculated to explore potential tampering. The correlation between accelerometer output and pedometer steps/day was strongest among participants in the WS group (r = 0.82, P ≤ 0.001), compared to the US (r = 0.63, P ≤ 0.001) and DS (r = 0.16, P = 0.324) groups. The DS (P ≤ 0.001) and US (P = 0.003), but not the WS (P = 0.891), groups showed evidence of reactivity. The results suggest that reactivity and tampering does occur in adolescents and contrary to existing research, pedometer monitoring protocols may influence participant behaviour.

Efficient purification of Apolipoprotein A1 (ApoA1) from plasma by HEA HyperCel™: An alternative approach.

PubMed

G, Arun Govind; Kamalanathan, Agamudi Shivasankaran; Vijayalakshmi, Mookambeswaran Arunachalam; Venkataraman, Krishnan

2018-01-15

HDL-ApoA1 plays a pivotal role in the prevention of atherosclerosis and cardiovascular diseases. ApoA1 purification from blood plasma has always remained tedious, involving multiple steps, large volumes of plasma and substantial loss in the final yield of pure ApoA1. In this study, a two-step method has been developed and optimized for the purification of ApoA1 from plasma. Plasma was first subjected to 60% ammonium sulphate (NH 4 ) 2 SO 4 precipitation and subsequently, ApoA1 was recovered using mixed mode chromatographic sorbent, HEA HyperCel™. ApoA1 was found to be enriched in 60% (NH 4 ) 2 SO 4 supernatant that was dialyzed and injected onto HEA sorbent with 50 mM phosphate buffer pH 7.4. The bound proteins were eluted by decreasing the pH in step-gradient from pH 7.4 to pH 4.0 and subsequently to pH 3.5 using 50 mM sodium acetate buffer. Gel electrophoresis showed elution of homogeneous apoA1 at pH 3.5, with purity and yield of 63%. An interesting feature of this approach is that the purified ApoA1 was monomeric with a mass of 28,079.30 Da as confirmed by MS analysis. This simple and efficient method of purification of apoA1 serves as an alternative method which can be combined with traditional approaches and has a great potential for biochemical and clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

A polyacrylamide-based silica stationary phase for the separation of carbohydrates using alcohols as the weak eluent in hydrophilic interaction liquid chromatography.

PubMed

Cai, Jianfeng; Cheng, Lingping; Zhao, Jianchao; Fu, Qing; Jin, Yu; Ke, Yanxiong; Liang, Xinmiao

2017-11-17

A hydrophilic interaction liquid chromatography (HILIC) stationary phase was prepared by a two-step synthesis method, immobilizing polyacrylamide on silica sphere particles. The stationary phase (named PA, 5μm dia) was evaluated using a mixture of carbohydrates in HILIC mode and the column efficiency reached 121,000Nm -1 . The retention behavior of carbohydrates on PA stationary phase was investigated with three different organic solvents (acetonitrile, ethanol and methanol) employed as the weak eluent. The strongest hydrophilicity of PA stationary phase was observed in both acetonitrile and methanol as the weak eluent, when compared with another two amide stationary phases. Attributing to its high hydrophilicity, three oligosaccharides (xylooligosaccharide, fructooligosaccharide and chitooligosaccharides) presented good retention on PA stationary phase using alcohols/water as mobile phase. Finally, PA stationary phase was successfully applied for the purification of galactooligosaccharides and saponins of Paris polyphylla. It is feasible to use safer and cheaper alcohols to replace acetonitrile as the weak eluent for green analysis and purification of polar compounds on PA stationary phase. Copyright © 2017. Published by Elsevier B.V.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

PubMed

Brito, Maíra M; Lúcio, Cristina F; Angrimani, Daniel S R; Losano, João Diego A; Dalmazzo, Andressa; Nichi, Marcílio; Vannucchi, Camila I

2017-01-02

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Purification of Active Myrosinase from Plants by Aqueous Two-Phase Counter-Current Chromatography

PubMed Central

Wade, Kristina L.; Ito, Yoichiro; Ramarathnam, Aarthi; Holtzclaw, W. David; Fahey, Jed W.

2014-01-01

Introduction Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (frombroccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (β-thioglucoside N-hydroxysulphate) precursors. Objective To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. Methods A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). Results Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. Conclusion Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable. PMID:25130502

Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.

PubMed

Wade, Kristina L; Ito, Yoichiro; Ramarathnam, Aarthi; Holtzclaw, W David; Fahey, Jed W

2015-01-01

Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (β-thioglucoside N-hydroxysulphate) precursors. To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable. Copyright © 2014 John Wiley & Sons, Ltd.

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

PubMed

Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

1999-01-01

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Polarization entanglement purification of nonlocal microwave photons based on the cross-Kerr effect in circuit QED

NASA Astrophysics Data System (ADS)

Zhang, Hao; Liu, Qian; Xu, Xu-Sheng; Xiong, Jun; Alsaedi, Ahmed; Hayat, Tasawar; Deng, Fu-Guo

2017-11-01

Microwave photons have become very important qubits in quantum communication, as the first quantum satellite has been launched successfully. Therefore, it is a necessary and meaningful task for ensuring the high security and efficiency of microwave-based quantum communication in practice. Here, we present an original polarization entanglement purification protocol for nonlocal microwave photons based on the cross-Kerr effect in circuit quantum electrodynamics (QED). Our protocol can solve the problem that the purity of maximally entangled states used for constructing quantum channels will decrease due to decoherence from environment noise. This task is accomplished by means of the polarization parity-check quantum nondemolition (QND) detector, the bit-flipping operation, and the linear microwave elements. The QND detector is composed of several cross-Kerr effect systems which can be realized by coupling two superconducting transmission line resonators to a superconducting molecule with the N -type level structure. We give the applicable experimental parameters of QND measurement system in circuit QED and analyze the fidelities. Our protocol has good applications in long-distance quantum communication assisted by microwave photons in the future, such as satellite quantum communication.

Quality by Design (QbD)-Based Process Development for Purification of a Biotherapeutic.

PubMed

Rathore, Anurag S

2016-05-01

Quality by Design (QbD) is currently receiving increased attention from the pharmaceutical community. As a result, most major biotech manufacturers are in varying stages of implementing QbD. Here, I present a case study that illustrates the step-by-step development using QbD of a purification process for the production of a biosimilar product: granulocyte colony-stimulating factor (GCSF). I also highlight and discuss the advantages that QbD-based process development offers over traditional approaches. The case study is intended to help those who wish to implement QbD towards the development and commercialization of biotech products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application.

PubMed

Vijay Simha, B; Sood, S K; Kumariya, Rashmi; Garsa, Anita Kumari

2012-10-12

The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20 g/l of glucose or lactose at 20 and 24 h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200 rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×10⁵ AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15 μg from 20 μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6 kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1. Copyright © 2012 Elsevier GmbH. All rights reserved.

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

PubMed Central

Williams, Roger H.; Ward, Elaine; McCartney, H. Alastair

2001-01-01

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting. PMID:11375150

Efficient and high yield isolation of myoblasts from skeletal muscle.

PubMed

Shahini, Aref; Vydiam, Kalyan; Choudhury, Debanik; Rajabian, Nika; Nguyen, Thy; Lei, Pedro; Andreadis, Stelios T

2018-05-24

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10-20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation of PCR quality microbial community DNA from heavily contaminated environments.

PubMed

Gunawardana, Manjula; Chang, Simon; Jimenez, Abraham; Holland-Moritz, Daniel; Holland-Moritz, Hannah; La Val, Taylor P; Lund, Craig; Mullen, Madeline; Olsen, John; Sztain, Terra A; Yoo, Jennifer; Moss, John A; Baum, Marc M

2014-07-01

Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology. Copyright © 2014 Elsevier B.V. All rights reserved.

One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer.

PubMed

Lee, Young-Tae; Gibbons, Garrett; Lee, Shirley Y; Nikolovska-Coleska, Zaneta; Dou, Yali

2015-06-01

We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

Arsanilic acid modified superparamagnetic iron oxide nanoparticles for Purification of alkaline phosphatase from hen's egg yolk.

PubMed

Farzi-Khajeh, Hamed; Safa, Kazem D; Dastmalchi, Siavoush

2017-09-01

Recent studies of magnetic carrier technology have focused on its applications in separation and purification technologies, due to easy separation of the target from the reaction medium by applying an external magnetic field. In the present study, Fe 3 O 4 superparamagnetic nanoparticles were prepared to utilize a chemical co-precipitation method, then the surfaces of the nanoparticles were modified with arsanilic acid derivatives which were used as the specific nanocarriers for the affinity purification of alkaline phosphatase from the hen's egg yolk. The six different types of magnetic nanocarriers with varied lengths of the linkers were obtained. All samples were characterized step by step and validated using FTIR, SEM, EDX, VSM and XRD analysis methods As the results were shown, the use of inflexible tags with long linkers on the surface of the nanocarrier could lead to better results for separation of alkaline phosphatase from the hen's egg yolk with 76.2% recovery and 1361.7-fold purification. The molecular weight of the purified alkaline phosphatase was estimated to be 68kDa by SDS-PAGE. The results of this study showed that the novel magnetic nanocarriers were capable of purifying alkaline phosphatase in a practically time and cost effective way. Copyright © 2017 Elsevier B.V. All rights reserved.

Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

PubMed Central

Palmer, Ira; Wingfield, Paul T.

2013-01-01

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine·HCl as in basic Protocol 2. A Support Protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE (UNIT 10.1). Other methods discussed in the Commentary section of this unit include the direct purification of polyhistidine-tagged proteins solubilized in guanidine·HCl, preparative removal of guanidine·HCl by reversed-phase chromatography as a prelude to protein folding, and the solubilization of inclusion bodies with anionic detergents. PMID:23151747

Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

PubMed Central

Palmer, Ira; Wingfield, Paul T.

2012-01-01

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine·HCl as in basic Protocol 2. A Support Protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE (UNIT 10.1). Other methods discussed in the Commentary section of this unit include the direct purification of polyhistidine-tagged proteins solubilized in guanidine·HCl, preparative removal of guanidine·HCl by reversed-phase chromatography as a prelude to protein folding, and the solubilization of inclusion bodies with anionic detergents. PMID:18429271

Production of Candida antaractica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell & Expression in Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production

USDA-ARS?s Scientific Manuscript database

A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was produced using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. The lycotoxin-1 (Lyt-1) C3 variant gene ORF was added in-frame with the CALB ORF to pote...

Waste water biological purification plants of dairy products industry and energy management

NASA Astrophysics Data System (ADS)

Stepanov, Sergey; Solkina, Olga; Stepanov, Alexander; Zhukova, Maria

2017-10-01

The paper presents results of engineering and economical comparison of waste water biological purification plants of dairy products industry. Three methods of purification are compared: traditional biological purification with the use of secondary clarifiers and afterpurification through granular-bed filters, biomembrane technology and physical-and-chemical treatment together with biomembrane technology for new construction conditions. The improvement of the biological purification technology using nitro-denitrification and membrane un-mixing of sludge mixture is a promising trend in this area. In these calculations, an energy management which is widely applied abroad was used. The descriptions of the three methods are illustrated with structural schemes. Costs of equipment and production areas are taken from manufacturers’ data. The research is aimed at an engineering and economical comparison of new constructions of waste water purification of dairy products industry. The experiment demonstrates advantages of biomembrane technology in waste water purification. This technology offers prospects of 122 million rubles cost saving during 25 years of operation when compared with of the technology of preparatory reagent flotation and of 13.7 million rubles cost saving compared to the option of traditional biological purification.

Recombinant Expression and Purification of the Shigella Translocator IpaB.

PubMed

Barta, Michael L; Adam, Philip R; Dickenson, Nicholas E

2017-01-01

Type III secretion systems (T3SS) are highly conserved virulence factors employed by a large number of pathogenic gram-negative bacteria. Like many T3SS translocators, recombinant expression of the hydrophobic Shigella protein IpaB requires the presence of its cognate chaperone IpgC. Chaperone-bound IpaB is maintained in a nonfunctional state, which has hampered in vitro studies aimed at understanding molecular structure and function of this important class of T3SS proteins. Herein, we describe an expression and purification protocol that utilizes mild detergents to produce highly purified, homogeneous IpaB of defined oligomeric states.

Applying high-throughput methods to develop a purification process for a highly glycosylated protein.

PubMed

Sanaie, Nooshafarin; Cecchini, Douglas; Pieracci, John

2012-10-01

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient synthesis of pure monotosylated beta-cyclodextrin and its dimers.

PubMed

Tripodo, Giuseppe; Wischke, Christian; Neffe, Axel T; Lendlein, Andreas

2013-11-15

6-O-Monotosyl-β-cyclodextrin (mono-Ts-βCD) is one of the most important intermediates in the production of substituted βCD. So far, performing the monotosylation reaction and, in particular, the purification steps was challenging, relied on toxic solvents, and resulted in long and expensive procedures at, importantly, low yields. Here, the reaction of cyclodextrin with p-toluenesulfonyl chloride in aqueous environment is described to obtain a highly pure mono-Ts-βCD, for which a single-step purification with a cation exchange resin was applied. With this synthetic route and purification, yields could be increased from typically <10-15% to 35%, and organic solvents could be avoided. As characterized by FTIR, mass spectrometry, elemental analysis, and NMR, mono-Ts-βCD was obtained with a molar purity of >98mol%. From mono-Ts-βCD, β-cyclodextrin dimers linked by ethylenediamine (bis-Et-βCD) were successfully prepared (yield 93%, purity 96mol%) in a one-step approach using an anion exchange resin to trap leaving groups that typically interfere in the reaction. This synthesis procedure with a direct collection of side-products may be a general strategy applicable for nucleophilic substitution of tosylated cyclodextrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Phospholipase A2 from Bothrops alternatus (víbora de la cruz) venom. Purification and some characteristic properties.

PubMed

Nisenbom, H E; Seki, C; Vidal, J C

1986-01-01

One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.

Removal of furan and phenolic compounds from simulated biomass hydrolysates by batch adsorption and continuous fixed-bed column adsorption methods.

PubMed

Lee, Sang Cheol; Park, Sunkyu

2016-09-01

It has been proposed to remove all potential inhibitors and sulfuric acid in biomass hydrolysates generated from dilute-acid pretreatment of biomass, based on three steps of sugar purification process. This study focused on its first step in which furan and phenolic compounds were selectively removed from the simulated hydrolysates using activated charcoal. Batch adsorption experiments demonstrated that the affinity of activated charcoal for each component was highest in the order of vanillic acid, 4-hydroxybenzoic acid, furfural, acetic acid, sulfuric acid, and xylose. The affinity of activated charcoal for furan and phenolic compounds proved to be significantly higher than that of the other three components. Four separation strategies were conducted with a combination of batch adsorption and continuous fixed-bed column adsorption methods. It was observed that xylose loss was negligible with near complete removal of furan and phenolic compounds, when at least one fixed-bed column adsorption was implemented in the strategy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

PubMed

Dündar, Halil; Atakay, Mehmet; Çelikbıçak, Ömür; Salih, Bekir; Bozoğlu, Faruk

2015-01-01

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Purification of Carbon Nanotubes: Alternative Methods

NASA Technical Reports Server (NTRS)

Files, Bradley; Scott, Carl; Gorelik, Olga; Nikolaev, Pasha; Hulse, Lou; Arepalli, Sivaram

2000-01-01

Traditional carbon nanotube purification process involves nitric acid refluxing and cross flow filtration using surfactant TritonX. This is believed to result in damage to nanotubes and surfactant residue on nanotube surface. Alternative purification procedures involving solvent extraction, thermal zone refining and nitric acid refiuxing are used in the current study. The effect of duration and type of solvent to dissolve impurities including fullerenes and P ACs (polyaromatic compounds) are monitored by nuclear magnetic reasonance, high performance liquid chromatography, and thermogravimetric analysis. Thermal zone refining yielded sample areas rich in nanotubes as seen by scanning electric microscopy. Refluxing in boiling nitric acid seem to improve the nanotube content. Different procedural steps are needed to purify samples produced by laser process compared to arc process. These alternative methods of nanotube purification will be presented along with results from supporting analytical techniques.

Studies on separation and purification of fission (99)Mo from neutron activated uranium aluminum alloy.

PubMed

Rao, Ankita; Kumar Sharma, Abhishek; Kumar, Pradeep; Charyulu, M M; Tomar, B S; Ramakumar, K L

2014-07-01

A new method has been developed for separation and purification of fission (99)Mo from neutron activated uranium-aluminum alloy. Alkali dissolution of the irradiated target (100mg) results in aluminum along with (99)Mo and a few fission products passing into solution, while most of the fission products, activation products and uranium remain undissolved. Subsequent purification steps involve precipitation of aluminum as Al(OH)3, iodine as AgI/AgIO3 and molybdenum as Mo-α-benzoin oxime. Ruthenium is separated by volatilization as RuO4 and final purification of (99)Mo was carried out using anion exchange method. The radiochemical yield of fission (99)Mo was found to be >80% and the purity of the product was in conformity with the international pharmacopoeia standards. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Influence of Surface Morphology and Diffraction Resolution of Canavalin Crystals

NASA Technical Reports Server (NTRS)

Plomp, M.; Thomas, B. R.; Day, J. S.; McPherson, A.; Chernov, A. A.; Malkin, A.

2003-01-01

Canavalin crystals grown from material purified and not purified by High Performance Liquid Chromatography were studied by atomic force microscopy and x-ray diffraction. After purification, resolution was improved from 2.55Angstroms to 2.22Angstroms and jagged isotropic spiral steps transformed into regular, well polygonized steps.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

PubMed

Herrera-Asmat, Omar; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos J; Guerra, Daniel G; Kireeva, Maria L

2017-06-01

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α 2 ββ'ω·σ A can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD. Published by Elsevier Inc.

Aqueous Chloride Operations Overview: Plutonium and Americium Purification/Recovery

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

Gardner, Kyle Shelton; Kimball, David Bryan; Skidmore, Bradley Evan

These are a set of slides intended for an information session as part of recruiting activities at Brigham Young University. It gives an overview of aqueous chloride operations, specifically on plutonium and americium purification/recovery. This presentation details the steps taken perform these processes, from plutonium size reduction, dissolution, solvent extraction, oxalate precipitation, to calcination. For americium recovery, it details the CLEAR (chloride extraction and actinide recovery) Line, oxalate precipitation and calcination.

Two-Step Vapor/Liquid/Solid Purification

NASA Technical Reports Server (NTRS)

Holland, L. R.

1986-01-01

Vertical distillation system combines in single operation advantages of multiple zone refining with those of distillation. Developed specifically to load Bridgman-Stockbarger (vertical-solidification) growth ampoules with ultrapure tellurium and cadmium, system, with suitable modifications, serves as material refiner. In first phase of purification process, ampoule heated to drive off absorbed volatiles. Second phase, evaporator heated to drive off volatiles in charge. Third phase, slowly descending heater causes distillation from evaporator to growing crystal in ampoule.

Algorithmic cooling in liquid-state nuclear magnetic resonance

NASA Astrophysics Data System (ADS)

Atia, Yosi; Elias, Yuval; Mor, Tal; Weinstein, Yossi

2016-01-01

Algorithmic cooling is a method that employs thermalization to increase qubit purification level; namely, it reduces the qubit system's entropy. We utilized gradient ascent pulse engineering, an optimal control algorithm, to implement algorithmic cooling in liquid-state nuclear magnetic resonance. Various cooling algorithms were applied onto the three qubits of C132-trichloroethylene, cooling the system beyond Shannon's entropy bound in several different ways. In particular, in one experiment a carbon qubit was cooled by a factor of 4.61. This work is a step towards potentially integrating tools of NMR quantum computing into in vivo magnetic-resonance spectroscopy.

What's Living in Your World?

ERIC Educational Resources Information Center

Powell, Karen; Stiller, John

2005-01-01

The average biotech catalog contains a dizzying array of kits offering tried-and-true protocols in molecular biology and biochemistry. Prepackaged experiences, ranging from DNA fingerprinting to protein purification, are now available to high school students. Although commercial kits designed for education provide excellent hands-on experiences in…

Role of memory errors in quantum repeaters

NASA Astrophysics Data System (ADS)

Hartmann, L.; Kraus, B.; Briegel, H.-J.; Dür, W.

2007-03-01

We investigate the influence of memory errors in the quantum repeater scheme for long-range quantum communication. We show that the communication distance is limited in standard operation mode due to memory errors resulting from unavoidable waiting times for classical signals. We show how to overcome these limitations by (i) improving local memory and (ii) introducing two operational modes of the quantum repeater. In both operational modes, the repeater is run blindly, i.e., without waiting for classical signals to arrive. In the first scheme, entanglement purification protocols based on one-way classical communication are used allowing to communicate over arbitrary distances. However, the error thresholds for noise in local control operations are very stringent. The second scheme makes use of entanglement purification protocols with two-way classical communication and inherits the favorable error thresholds of the repeater run in standard mode. One can increase the possible communication distance by an order of magnitude with reasonable overhead in physical resources. We outline the architecture of a quantum repeater that can possibly ensure intercontinental quantum communication.

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

PubMed

Ritterhoff, Tobias; Das, Hrishikesh; Hao, Yuqing; Sakin, Volkan; Flotho, Annette; Werner, Andreas; Melchior, Frauke

2016-01-01

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.

PubMed

Sousa, A; Almeida, A M; Černigoj, U; Sousa, F; Queiroz, J A

2014-08-15

Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level. Copyright © 2014. Published by Elsevier B.V.

Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

PubMed Central

Casadonte, Rita; Caprioli, Richard M

2012-01-01

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets.

PubMed

Struwe, Weston B; Agravat, Sanjay; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P; Costello, Catherine E; Dell, Anne; Ten Feizi; Haslam, Stuart M; Karlsson, Niclas G; Khoo, Kay-Hooi; Kolarich, Daniel; Liu, Yan; McBride, Ryan; Novotny, Milos V; Packer, Nicolle H; Paulson, James C; Rapp, Erdmann; Ranzinger, Rene; Rudd, Pauline M; Smith, David F; Tiemeyer, Michael; Wells, Lance; York, William S; Zaia, Joseph; Kettner, Carsten

2016-09-01

The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Synthesis of 4'-Selenoribonucleosides, the Building Blocks of 4'-SelenoRNA, Using a Hypervalent Iodine.

PubMed

Saito-Tarashima, Noriko; Ota, Masashi; Minakawa, Noriaki

2017-09-18

Herein is described a detailed protocol for the synthesis of 4'-selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4'-selenoRNA. Large-scale synthesis of a 4-selenosugar starting from D-ribose is achieved in eight steps, including a final chromatographic purification. The resulting 4-selenosugar is then subjected to the one-pot Pummerer-like reaction using hypervalent iodine in the presence of silylated nucleobases. The reaction with silylated uracil affords the desired 4'-selenouridine derivatives with excellent β-selectivity and in good yield. Conversely, when purine nucleobases are used in the Pummerer-like reaction, N7 4'-selenoribonucleoside isomers are obtained alongside the desired N9 isomers. However, the undesired N7 isomers can be converted to the desired N9 ones under acidic conditions. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

PubMed

McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

2016-08-06

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

PubMed

Stressler, Timo; Tanzer, Coralie; Ewert, Jacob; Claaßen, Wolfgang; Fischer, Lutz

2017-03-01

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His 6 -tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His 6 -tag harboring PepA; the K M value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

Experimental study and simulation of phosphorus purification effects of bioretention systems on urban surface runoff

PubMed Central

Liang, Zheng; Li, Yajiao; Li, Peng; Jiang, Chunbo

2018-01-01

Excessive phosphorus (P) contributes to eutrophication by degrading water quality and limiting human use of water resources. Identifying economic and convenient methods to control soluble reactive phosphorus (SRP) pollution in urban runoff is the key point of rainwater management strategies. Through three series of different tests involving influencing factors, continuous operation and intermittent operation, this study explored the purification effects of bioretention tanks under different experimental conditions, it included nine intermittent tests, single field continuous test with three groups of different fillers (Fly ash mixed with sand, Blast furnace slag, and Soil), and eight intermittent tests with single filler (Blast furnace slag mixed with sand). Among the three filler combinations studied, the filler with fly ash mixed with sand achieved the best pollution reduction efficiency. The setting of the submerged zone exerted minimal influence on the P removal of the three filler combinations. An extension of the dry period slightly promoted the P purification effect. The combination of fly ash mixed with sand demonstrated a positive purification effect on SRP during short- or long-term simulated rainfall duration. Blast furnace slag also presented a positive purification effect in the short term, although its continuous purification effect on SRP was poor in the long term. The purification abilities of soil in the short and long terms were weak. Under intermittent operations across different seasons, SRP removal was unstable, and effluent concentration processes were different. The purification effect of the bioretention system on SRP was predicted through partial least squares regression (PLS) modeling analysis. The event mean concentration removal of SRP was positively related to the adsorption capacity of filler and rainfall interval time and negatively related to submerged zones, influent concentration and volume. PMID:29742120

Practical State Machine Replication with Confidentiality

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

Duan, Sisi; Zhang, Haibin

2016-01-01

We study how to enable arbitrary randomized algorithms in Byzantine fault-tolerant (BFT) settings. We formalize a randomized BFT protocol and provide a simple and efficient construction that can be built on any existing BFT protocols while adding practically no overhead. We go one step further to revisit a confidential BFT protocol (Yin et al., SOSP '03). We show that their scheme is potentially susceptible to safety and confidentiality attacks. We then present a new protocol that is secure in the stronger model we formalize, by extending the idea of a randomized BFT protocol. Our protocol uses only efficient symmetric cryptography,more » while Yin et al.'s uses costly threshold signatures. We implemented and evaluated our protocols on microbenchmarks and real-world use cases. We show that our randomized BFT protocol is as efficient as conventional BFT protocols, and our confidential BFT protocol is two to three orders of magnitude faster than Yin et al.'s, which is less secure than ours.« less

A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

PubMed

Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

2015-07-24

An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

PubMed

Buyel, Johannes F; Hubbuch, Jürgen; Fischer, Rainer

2016-08-08

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

PubMed Central

Rodrigo, Lourdes; Gil, Fernando; Hernandez, Antonio F; Lopez, Olga; Pla, Antonio

2003-01-01

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability. PMID:12946270

Preparative isolation and purification of three sesquiterpenoid lactones from Eupatorium lindleyanum DC. by high-speed counter-current chromatography.

PubMed

Yan, Guilong; Ji, Lilian; Luo, Yuming; Hu, Yonghong

2012-07-27

A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from Eupatorium lindleyanum DC. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:4:2:3, v/v/v/v) was selected. From 540 mg of the n-butanol fraction of Eupatorium lindleyanum DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxytigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and ¹H-NMR.

Postextraction Separation, On-Board Storage, and Catalytic Conversion of Methane in Natural Gas: A Review.

PubMed

Saha, Dipendu; Grappe, Hippolyte A; Chakraborty, Amlan; Orkoulas, Gerassimos

2016-10-12

In today's perspective, natural gas has gained considerable attention, due to its low emission, indigenous availability, and improvement in the extraction technology. Upon extraction, it undergoes several purification protocols including dehydration, sweetening, and inert rejection. Although purification is a commercially established technology, several drawbacks of the current process provide an essential impetus for developing newer separation protocols, most importantly, adsorption and membrane separation. This Review summarizes the needs of natural gas separation, gives an overview of the current technology, and provides a detailed discussion of the progress in research on separation and purification of natural gas including the benefits and drawbacks of each of the processes. The transportation sector is another growing sector of natural gas utilization, and it requires an efficient and safe on-board storage system. Compressed natural gas (CNG) and liquefied natural gas (LNG) are the most common forms in which natural gas can be stored. Adsorbed natural gas (ANG) is an alternate storage system of natural gas, which is advantageous as compared to CNG and LNG in terms of safety and also in terms of temperature and pressure requirements. This Review provides a detailed discussion on ANG along with computation predictions. The catalytic conversion of methane to different useful chemicals including syngas, methanol, formaldehyde, dimethyl ether, heavier hydrocarbons, aromatics, and hydrogen is also reviewed. Finally, direct utilization of methane onto fuel cells is also discussed.

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

PubMed

Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

2014-06-01

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

PubMed

Kelley, Brian D; Tobler, Scott A; Brown, Paul; Coffman, Jonathan L; Godavarti, Ranga; Iskra, Timothy; Switzer, Mary; Vunnum, Suresh

2008-10-15

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved. (c) 2008 Wiley Periodicals, Inc.

Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

PubMed Central

Alibolandi, Mona; Mirzahoseini, Hasan

2011-01-01

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. PMID:21837279

Extraction and purification methods in downstream processing of plant-based recombinant proteins.

PubMed

Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz

2016-04-01

During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described. Copyright © 2015 Elsevier Inc. All rights reserved.

Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

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

Mao, Xun; Liu, Guodong; Wang, Shengfu

2008-11-01

We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale.

PubMed

O'Mahony, Kevin; Freitag, Ruth; Hilbrig, Frank; Müller, Patrick; Schumacher, Ivo

2005-09-23

The paper addresses the question of how to achieve bacterial lysis in large-scale plasmid DNA production processes, where conventional alkaline lysis may become awkward to handle. Bacteria were grown in shaker flasks and a bioreactor. Suboptimal growth conditions were found advantageous for stable plasmid production at high copy numbers (up to 25mg/L could be achieved). Cells were harvested by filtration in the presence of a filter aid. A linear relationship between the biomass and the optimal filter aid concentration in terms of back pressure could be established. Bacteria-containing filter cakes were washed with isotonic buffer and lysis was achieved in situ by a two-step protocol calling for fragilisation of the cells followed by heat lysis in a suitable buffer. RNA and other soluble cell components where washed out of the cake during this step, while the plasmid DNA was retained. Afterwards a clear lysate containing relatively pure plasmid DNA could be eluted from the cake mostly as the desired supercoiled topoisomer, while cell debris and genomic DNA were retained. Lysis is, thus, integrated not only with cell capture but also with a significant degree of isolation/purification, as most impurities were considerably reduced during the procedure.

Desensitization to rituximab in a multidisciplinary setting.

PubMed

Amorós-Reboredo, Patrícia; Sánchez-López, Jaime; Bastida-Fernández, Carla; do Pazo-Oubiña, Fernando; Borràs-Maixenchs, Núria; Giné, Eva; Valero, Antonio; Creus-Baró, Natàlia

2015-10-01

The need to offer first-line therapy to the increasing number of patients who have suffered an hypersensitivity reaction has stimulated the use of rapid desensitization protocols. To present our experience working as a multidisciplinary team using a rituximab rapid desensitization scheme. Patient demographics, allergic reaction, skin tests to rituximab, number of desensitizations, reactions during the desensitization protocol and actions taken, number of administered and completed cycles, were retrospectively collected in patients who received at least one desensitization to rituximab. Number of desensitizations successfully managed. Between 2012 and June 2013 five patients received a total of 19 desensitizations to rituximab using a 12 step rapid desensitization protocol. All patients received the scheduled chemotherapeutic cycles as inpatients, with no delay in administration dates. Three patients presented a hypersensitivity reaction during the first desensitization and in one patient the event occurred again during the second treatment cycle. All reactions occurred in the last step, when the infusion rate reached the maximum speed. The developed protocol for rapid desensitization was successful in five patients receiving rituximab. Patients could receive the full intended dose.

Succinonitrile Purification Facility

NASA Technical Reports Server (NTRS)

2003-01-01

The Succinonitrile (SCN) Purification Facility provides succinonitrile and succinonitrile alloys to several NRA selected investigations for flight and ground research at various levels of purity. The purification process employed includes both distillation and zone refining. Once the appropriate purification process is completed, samples are characterized to determine the liquidus and/or solidus temperature, which is then related to sample purity. The lab has various methods for measuring these temperatures with accuracies in the milliKelvin to tenths of milliKelvin range. The ultra-pure SCN produced in our facility is indistinguishable from the standard material provided by NIST to well within the stated +/- 1.5mK of the NIST triple point cells. In addition to delivering material to various investigations, our current activities include process improvement, characterization of impurities and triple point cell design and development. The purification process is being evaluated for each of the four vendors to determine the efficacy of each purification step. We are also collecting samples of the remainder from distillation and zone refining for analysis of the constituent impurities. The large triple point cells developed will contain SCN with a melting point of 58.0642 C +/- 1.5mK for use as a calibration standard for Standard Platinum Resistance Thermometers (SPRTs).

Process of electrolysis and fractional crystallization for aluminum purification

DOEpatents

Dawless, R.K.; Bowman, K.A.; Mazgaj, R.M.; Cochran, C.N.

1983-10-25

A method is described for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm. 2 figs.

Process of electrolysis and fractional crystallization for aluminum purification

DOEpatents

Dawless, Robert K.; Bowman, Kenneth A.; Mazgaj, Robert M.; Cochran, C. Norman

1983-10-25

A method for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm.

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

PubMed

Kim, Sung Hoon; Jeyakumar, M; Katzenellenbogen, John A

2007-10-31

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases.

PubMed

Xin, Huaxia; Dai, Zhuoshun; Cai, Jianfeng; Ke, Yanxiong; Shi, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao

2017-08-04

Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

PubMed

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Effect of additives on the purification of urease

NASA Astrophysics Data System (ADS)

Yu, X.; Wang, J.; Ulrich, J.

2015-12-01

The effect of additives on the purification of proteins was investigated. The target protein studied here is the enzyme urease. Studies on the purification of urease from jack bean meal were carried out. 32% (v/v) acetone was utilized to extract urease from the jack bean meal. Further purification by crystallization with the addition of 2-mercaptoethanol and EDTA disodium salt dehydrate was carried out. It was found out that the presence of additives can affect the selectivity of the crystallization. Increases in both purity and yield of the urease after crystallization were observed in the presence of additives, which were proven using both SDS-PAGE and activity. Urease crystals with a yield of 69.9% and a purity of 85.1% were obtained in one crystallization step in the presence of additives. Furthermore, the effect of additives on the thermodynamics and kinetics of urease crystallization was studied.

Purification process for vertically aligned carbon nanofibers

NASA Technical Reports Server (NTRS)

Nguyen, Cattien V.; Delziet, Lance; Matthews, Kristopher; Chen, Bin; Meyyappan, M.

2003-01-01

Individual, free-standing, vertically aligned multiwall carbon nanotubes or nanofibers are ideal for sensor and electrode applications. Our plasma-enhanced chemical vapor deposition techniques for producing free-standing and vertically aligned carbon nanofibers use catalyst particles at the tip of the fiber. Here we present a simple purification process for the removal of iron catalyst particles at the tip of vertically aligned carbon nanofibers derived by plasma-enhanced chemical vapor deposition. The first step involves thermal oxidation in air, at temperatures of 200-400 degrees C, resulting in the physical swelling of the iron particles from the formation of iron oxide. Subsequently, the complete removal of the iron oxide particles is achieved with diluted acid (12% HCl). The purification process appears to be very efficient at removing all of the iron catalyst particles. Electron microscopy images and Raman spectroscopy data indicate that the purification process does not damage the graphitic structure of the nanotubes.

Magnetic particles as powerful purification tool for high sensitive mass spectrometric screening procedures.

PubMed

Peter, Jochen F; Otto, Angela M

2010-02-01

The effective isolation and purification of proteins from biological fluids is the most crucial step for a successful protein analysis when only minute amounts are available. While conventional purification methods such as dialysis, ultrafiltration or protein precipitation often lead to a marked loss of protein, SPE with small-sized particles is a powerful alternative. The implementation of particles with superparamagnetic cores facilitates the handling of those particles and allows the application of particles in the nanometer to low micrometer range. Due to the small diameters, magnetic particles are advantageous for increasing sensitivity when using subsequent MS analysis or gel electrophoresis. In the last years, different types of magnetic particles were developed for specific protein purification purposes followed by analysis or screening procedures using MS or SDS gel electrophoresis. In this review, the use of magnetic particles for different applications, such as, the extraction and analysis of DNA/RNA, peptides and proteins, is described.

A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

PubMed

Hailemariam, Zerihun; Ahmed, Jabbar Sabir; Clausen, Peter-Henning; Nijhof, Ard Menzo

2017-01-01

An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic ® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex ® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent. Copyright © 2016 Elsevier GmbH. All rights reserved.

[Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

PubMed

Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

2012-01-01

Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.

Preparation of GST Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

PubMed

Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

2017-01-01

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Clomiphene citrate 'stair-step' protocol vs. traditional protocol in patients with polycystic ovary syndrome: a randomized controlled trial.

PubMed

Deveci, Canan Dura; Demir, Berfu; Sengul, Ozlem; Dilbaz, Berna; Goktolga, Umit

2015-01-01

To evaluate the efficacy of the stair-step protocol using clomiphene citrate (CC) and to assess the uterine and systemic side effects in patients with polycystic ovary syndrome (PCOS). A total of 60 PCOS patients who failed to respond to 50 mg/day for 5 days of CC treatment within the cycle were randomly allocated to the control (traditional protocol) and study (stair-step protocol) groups. In the stair-step protocol,patients were treated with CC 50 mg/day for 5 days and then in nonresponsive patients, the dosage was increased to 100 mg/day for 5 days in the same cycle. Patients who failed the 50 mg/day CC treatment in the previous cycle were stimulated with 100 mg/day CC and were accepted as the control group. Ovulation and pregnancy rates, duration of treatment and uterine and systemic side effects were evaluated. Ovulation and pregnancy rates were similar between the stair-step and the control group (43.3 vs. 33.3 %, respectively) (16.7 vs. 10 %, respectively). The duration of treatment was significantly shorter in stair-step compared to traditional protocol (20.5 ± 2.0 vs. 48.6 ± 2.4 days, respectively). There were no significant differences in the systemic side effects between the groups. Uterine side effects were evaluated with endometrial thickness and uterine artery Doppler ultrasound; no significant differences were observed in stair-step compared to traditional protocol. The stair-step protocol was determined to have a significantly shorter treatment period without any detrimental effect on the ovulation and pregnancy rates.

One-step purification of a functional, constitutively activated form of visual arrestin.

PubMed

Huang, Li; Mao, Xiang; Abdulaev, Najmoutin G; Ngo, Tony; Liu, Wei; Ridge, Kevin D

2012-03-01

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗). The results show that both prodomain/Profinity eXact™ fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale™ Mini Profinity eXact™ cartridge on the Profinia™ purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho∗. Copyright © 2011 Elsevier Inc. 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 blood purification: mixed-matrix membranes combining diffusion and adsorption in one step.

PubMed

Tijink, Marlon S L; Wester, Maarten; Sun, Junfen; Saris, Anno; Bolhuis-Versteeg, Lydia A M; Saiful, Saiful; Joles, Jaap A; Borneman, Zandrie; Wessling, Matthias; Stamatialis, Dimitris F

2012-07-01

Hemodialysis is a commonly used blood purification technique in patients requiring kidney replacement therapy. Sorbents could increase uremic retention solute removal efficiency but, because of poor biocompatibility, their use is often limited to the treatment of patients with acute poisoning. This paper proposes a novel membrane concept for combining diffusion and adsorption of uremic retention solutes in one step: the so-called mixed-matrix membrane (MMM). In this concept, adsorptive particles are incorporated in a macro-porous membrane layer whereas an extra particle-free membrane layer is introduced on the blood-contacting side of the membrane to improve hemocompatibility and prevent particle release. These dual-layer mixed-matrix membranes have high clean-water permeance and high creatinine adsorption from creatinine model solutions. In human plasma, the removal of creatinine and of the protein-bound solute para-aminohippuric acid (PAH) by single and dual-layer membranes is in agreement with the removal achieved by the activated carbon particles alone, showing that under these experimental conditions the accessibility of the particles in the MMM is excellent. This study proves that the combination of diffusion and adsorption in a single step is possible and paves the way for the development of more efficient blood purification devices, excellently combining the advantages of both techniques. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Extraction chromatographic separations of tantalum and tungsten from hafnium and complex matrix constituents

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

Snow, Mathew S.; Finck, Martha R.; Carney, Kevin P.

2017-02-01

Ta, Hf, and W analyses from complex matrices (including environmental samples) require high purification of these analytes from each other and major/trace matrix constituents, however, current state-of-the-art Ta/Hf/W separations rely on traditional anion exchange approaches that suffer from relatively similar distribution coefficient (Kd) values for these analytes. This work reports assessment of three commercially available extraction chromatographic resins (TEVA, TRU, and UTEVA) for Ta/Hf/W separations. Batch contact studies show differences in Ta/W,Hf Kd values of up to 106, representing an improvement of a factor of 100 and 300 in Ta/Hf and Ta/W Kd values (respectively) over AG1x4 resin. Variations inmore » the Kd values as a function of HCl concentration for TRU resin show that this resin is well suited for Ta/Hf/W separations, with Ta/Hf, Ta/W, and W/Hf Kd value improvements of 10, 200, and 30 (respectively) over AG1x4 resin. Finally, analyses of digested soil samples (NIST 2710a) using TRU resin and tandem TEVA-TRU columns demonstrate the ability to achieve extremely high purification (>99%) of Ta and W from each other and Hf, as well as enabling very high purification of Ta and W from the major and trace elemental constituents present in soils, using a single chromatographic step.« less

Extraction chromatographic separations of tantalum and tungsten from hafnium and complex matrix constituents

DOE PAGES

Snow, Mathew S.; Finck, Martha R.; Carney, Kevin P.; ...

2017-01-08

Ta, Hf, and W analyses from complex matrices (including environmental samples) require high purification of these analytes from each other and major/trace matrix constituents, but, current state-of-the-art Ta/Hf/W separations rely on traditional anion exchange approaches that suffer from relatively similar distribution coefficient (Kd) values for these analytes. Our work reports assessment of three commercially available extraction chromatographic resins (TEVA, TRU, and UTEVA) for Ta/Hf/W separations. Batch contact studies show differences in Ta/W,Hf Kd values of up to 10 6, representing an improvement of a factor of 100 and 300 in Ta/Hf and Ta/W Kd values (respectively) over AG1x4 resin. Furthermore,more » variations in the Kd values as a function of HCl concentration for TRU resin show that this resin is well suited for Ta/Hf/W separations, with Ta/Hf, Ta/W, and W/Hf Kd value improvements of 10, 200, and 30 (respectively) over AG1x4 resin. Finally, analyses of digested soil samples (NIST 2710a) using TRU resin and tandem TEVA-TRU columns demonstrate the ability to achieve extremely high purification (>99%) of Ta and W from each other and Hf, as well as enabling very high purification of Ta and W from the major and trace elemental constituents present in soils, using a single chromatographic step.« less

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

Preparative separation and purification of epigallocatechin gallate from green tea extracts using a silica adsorbent containing β-cyclodextrin.

PubMed

Lai, Shih-Ming; Gu, Jhe-Yu; Huang, Bing-Hao; Chang, Chieh-Ming J; Lee, Wen-Lung

2012-03-01

A silica adsorbent containing β-cyclodextrin (β-CD) has been developed and used for the separation and purification of epigallocatechin gallate (EGCG) from the green tea extracts. The batch adsorption experiments demonstrated that, the β-CD bonded silica adsorbent possessed excellent adsorption equilibrium capacity (> 55 mg/g adsorbent) and adsorption ratio (>95%) for EGCG compared to the other tea catechins and caffeine. The excellent adsorption capacity and selectivity for EGCG are attributed to the specific interactions between β-CD and EGCG. The preparative separation and purification performance of EGCG on the β-CD bonded silica column (220 mm L × 15 mm i.d., 40-63 μm) was then evaluated. The column was operated in the polar organic mode using methanol/acetonitrile/acetic acid as the mobile phase and eluted under a three-step gradient elution program. The sample was dissolved in acetonitrile and loaded on a preparative scale of about 0.8 mg/g adsorbent. Under the optimal chromatographic conditions, the target compound, EGCG, being the most retained species, was obtained at a purity of about 90% with a recovery of about 90%. The productivity of EGCG was about 6 mg per injection, which can be further increased by scaling-up the chromatographic system. Copyright © 2012 Elsevier B.V. All rights reserved.

Discussion on runoff purification technology of highway bridge deck based on water quality safety

NASA Astrophysics Data System (ADS)

Tan, Sheng-guang; Liu, Xue-xin; Zou, Guo-ping; Xiong, Xin-zhu; Tao, Shuang-cheng

2018-06-01

Aiming at the actual problems existing, including a poor purification effect of highway bridge runoff collection and treatment system across sensitive water and necessary manual emergency operation, three kinds of technology, three pools system of bridge runoff purification, the integral pool of bridge runoff purification and ecological planting tank, are put forward by optimizing the structure of purification unit and system setting. At the same time, we come up with an emergency strategy for hazardous material leakage basing on automatic identification and remote control of traffic accidents. On the basis of combining these with the optimized pool structure, sensitive water safety can be guaranteed and water pollution, from directly discharging of bridge runoff, can be decreased. For making up for the shortages of green highway construction technology, the technique has important reference value.

Orofacial lymphatic malformation: management with a three steps diode laser protocol

NASA Astrophysics Data System (ADS)

Miccoli, Simona; Tempesta, Angela; Limongelli, Luisa; Caporusso, Concetta; Di Venere, Daniela; Petruzzi, Massimo; Lacaita, Mariagrazia; Maiorano, Eugenio; Favia, Gianfranco

2014-01-01

Lymphatic Malformation (LM) according to ISSVA Classification, is a rare benign disorder with unknown aetiology. LM may grow slowly over years or develop rapidly over the course of days becoming a bulky lump, infected or bleeding. We propose our three steps Diode Laser protocol for LM management, based on its persistent vascular blood component. 1. Histological and cytological examination, to evaluate the vascular blood component (10-40%), shows mature lymphocytes with red blood cells and endothelial cells. 2. Diode Laser Photocoagulation (DLP) in pulsed mode (on 100ms / off 400ms) at 10W and 800nm with a 300μm fibre kept 2-3mm from the tissues, to reduce the lesion. 3. Diode Laser surgical excision in pulsed mode (on 50ms / off 200ms) at 8W and 800nm with a 300 μm fibre in close contact with tissues, and histological intraoperative margins control on frozen sections. Even if it has inconstant results (lesions decreasing rate is 10% to 40% proportionally to vascular blood component), DLP simplifies the last and the most important step. Use of Diode Laser also in surgical excision reduces intra and postoperatory complications.

One Step Quantum Key Distribution Based on EPR Entanglement.

PubMed

Li, Jian; Li, Na; Li, Lei-Lei; Wang, Tao

2016-06-30

A novel quantum key distribution protocol is presented, based on entanglement and dense coding and allowing asymptotically secure key distribution. Considering the storage time limit of quantum bits, a grouping quantum key distribution protocol is proposed, which overcomes the vulnerability of first protocol and improves the maneuverability. Moreover, a security analysis is given and a simple type of eavesdropper's attack would introduce at least an error rate of 46.875%. Compared with the "Ping-pong" protocol involving two steps, the proposed protocol does not need to store the qubit and only involves one step.

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

PubMed

Hartmann, B M; Kaar, W; Yoo, I K; Lua, L H L; Falconer, R J; Middelberg, A P J

2009-12-01

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

Step Detection and Activity Recognition Accuracy of Seven Physical Activity Monitors

PubMed Central

Storm, Fabio A.; Heller, Ben W.; Mazzà, Claudia

2015-01-01

The aim of this study was to compare the seven following commercially available activity monitors in terms of step count detection accuracy: Movemonitor (Mc Roberts), Up (Jawbone), One (Fitbit), ActivPAL (PAL Technologies Ltd.), Nike+ Fuelband (Nike Inc.), Tractivity (Kineteks Corp.) and Sensewear Armband Mini (Bodymedia). Sixteen healthy adults consented to take part in the study. The experimental protocol included walking along an indoor straight walkway, descending and ascending 24 steps, free outdoor walking and free indoor walking. These tasks were repeated at three self-selected walking speeds. Angular velocity signals collected at both shanks using two wireless inertial measurement units (OPAL, ADPM Inc) were used as a reference for the step count, computed using previously validated algorithms. Step detection accuracy was assessed using the mean absolute percentage error computed for each sensor. The Movemonitor and the ActivPAL were also tested within a nine-minute activity recognition protocol, during which the participants performed a set of complex tasks. Posture classifications were obtained from the two monitors and expressed as a percentage of the total task duration. The Movemonitor, One, ActivPAL, Nike+ Fuelband and Sensewear Armband Mini underestimated the number of steps in all the observed walking speeds, whereas the Tractivity significantly overestimated step count. The Movemonitor was the best performing sensor, with an error lower than 2% at all speeds and the smallest error obtained in the outdoor walking. The activity recognition protocol showed that the Movemonitor performed best in the walking recognition, but had difficulty in discriminating between standing and sitting. Results of this study can be used to inform choice of a monitor for specific applications. PMID:25789630

Step detection and activity recognition accuracy of seven physical activity monitors.

PubMed

Storm, Fabio A; Heller, Ben W; Mazzà, Claudia

2015-01-01

The aim of this study was to compare the seven following commercially available activity monitors in terms of step count detection accuracy: Movemonitor (Mc Roberts), Up (Jawbone), One (Fitbit), ActivPAL (PAL Technologies Ltd.), Nike+ Fuelband (Nike Inc.), Tractivity (Kineteks Corp.) and Sensewear Armband Mini (Bodymedia). Sixteen healthy adults consented to take part in the study. The experimental protocol included walking along an indoor straight walkway, descending and ascending 24 steps, free outdoor walking and free indoor walking. These tasks were repeated at three self-selected walking speeds. Angular velocity signals collected at both shanks using two wireless inertial measurement units (OPAL, ADPM Inc) were used as a reference for the step count, computed using previously validated algorithms. Step detection accuracy was assessed using the mean absolute percentage error computed for each sensor. The Movemonitor and the ActivPAL were also tested within a nine-minute activity recognition protocol, during which the participants performed a set of complex tasks. Posture classifications were obtained from the two monitors and expressed as a percentage of the total task duration. The Movemonitor, One, ActivPAL, Nike+ Fuelband and Sensewear Armband Mini underestimated the number of steps in all the observed walking speeds, whereas the Tractivity significantly overestimated step count. The Movemonitor was the best performing sensor, with an error lower than 2% at all speeds and the smallest error obtained in the outdoor walking. The activity recognition protocol showed that the Movemonitor performed best in the walking recognition, but had difficulty in discriminating between standing and sitting. Results of this study can be used to inform choice of a monitor for specific applications.

Fluidic Manufacture of Star-Shaped Gold Nanoparticles.

PubMed

Silvestri, Alessandro; Lay, Luigi; Psaro, Rinaldo; Polito, Laura; Evangelisti, Claudio

2017-07-21

Star-shaped gold nanoparticles (StarAuNPs) are extremely attractive nanomaterials, characterized by localized surface plasmon resonance which could be potentially employed in a large number of applications. However, the lack of a reliable and reproducible synthetic protocols for the production of StarAuNPs is the major limitation to their spreading. For the first time, here we present a robust protocol to manufacture reproducible StarAuNPs by exploiting a fluidic approach. Star-shaped AuNPs have been synthesized by means of a seed-less protocol, employing ascorbic acid as reducing agent at room temperature. Moreover, the versatility of the bench-top microfluidic protocol has been exploited to afford hydrophilic, hydrophobic and solid-supported engineered StarAuNPs, by avoiding intermediate NP purifications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

PubMed

Sanz, J M; Lopez, R; Garcia, J L

1988-05-23

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements.

PubMed

Gjoka, Xhorxhi; Gantier, Rene; Schofield, Mark

2017-01-20

The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25L volume of harvested cell culture fluid (HCCF) in less than 12h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Optimal setups for forced-choice staircases with fixed step sizes.

PubMed

García-Pérez, M A

2000-01-01

Forced-choice staircases with fixed step sizes are used in a variety of formats whose relative merits have never been studied. This paper presents a comparative study aimed at determining their optimal format. Factors included in the study were the up/down rule, the length (number of reversals), and the size of the steps. The study also addressed the issue of whether a protocol involving three staircases running for N reversals each (with a subsequent average of the estimates provided by each individual staircase) has better statistical properties than an alternative protocol involving a single staircase running for 3N reversals. In all cases the size of a step up was different from that of a step down, in the appropriate ratio determined by García-Pérez (Vision Research, 1998, 38, 1861 - 1881). The results of a simulation study indicate that a) there are no conditions in which the 1-down/1-up rule is advisable; b) different combinations of up/down rule and number of reversals appear equivalent in terms of precision and cost: c) using a single long staircase with 3N reversals is more efficient than running three staircases with N reversals each: d) to avoid bias and attain sufficient accuracy, threshold estimates should be based on at least 30 reversals: and e) to avoid excessive cost and imprecision, the size of the step up should be between 2/3 and 3/3 the (known or presumed) spread of the psychometric function. An empirical study with human subjects confirmed the major characteristics revealed by the simulations.

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.

PubMed

Xu, Zhihao; Li, Jason; Zhou, Joe X

2012-01-01

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.

Purification and characterization of midgut α-amylase in a predatory bug, Andralus spinidens

PubMed Central

Sorkhabi-Abdolmaleki, Sahar; Zibaee, Arash; Hoda, Hassan; FazeliDinan, Mahmoud

2014-01-01

Abstract α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch. PMID:25373212

High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase.

PubMed

Bordeaux, Mélanie; de Girval, Diane; Rullaud, Robin; Subileau, Maeva; Dubreucq, Eric; Drone, Jullien

2014-01-01

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.

Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis.

PubMed

Qi, Xiao-Hua; Zhang, Li-Wei; Zhang, Xin-Xiang

2008-08-01

A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.

PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

PubMed

Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

2014-01-01

X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed 'PredPPCrys' using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of currently non-crystallizable proteins were provided as compendium data, which are anticipated to facilitate target selection and design for the worldwide structural genomics consortium. PredPPCrys is freely available at http://www.structbioinfor.org/PredPPCrys.

Methodology for the specification of communication activities within the framework of a multi-layered architecture: Toward the definition of a knowledge base

NASA Astrophysics Data System (ADS)

Amyay, Omar

A method defined in terms of synthesis and verification steps is presented. The specification of the services and protocols of communication within a multilayered architecture of the Open Systems Interconnection (OSI) type is an essential issue for the design of computer networks. The aim is to obtain an operational specification of the protocol service couple of a given layer. Planning synthesis and verification steps constitute a specification trajectory. The latter is based on the progressive integration of the 'initial data' constraints and verification of the specification originating from each synthesis step, through validity constraints that characterize an admissible solution. Two types of trajectories are proposed according to the style of the initial specification of the service protocol couple: operational type and service supplier viewpoint; knowledge property oriented type and service viewpoint. Synthesis and verification activities were developed and formalized in terms of labeled transition systems, temporal logic and epistemic logic. The originality of the second specification trajectory and the use of the epistemic logic are shown. An 'artificial intelligence' approach enables a conceptual model to be defined for a knowledge base system for implementing the method proposed. It is structured in three levels of representation of the knowledge relating to the domain, the reasoning characterizing synthesis and verification activities and the planning of the steps of a specification trajectory.

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.

An overview of the kinetic parameters of class B beta-lactamases.

PubMed Central

Felici, A; Amicosante, G; Oratore, A; Strom, R; Ledent, P; Joris, B; Fanuel, L; Frère, J M

1993-01-01

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column. Images Figure 1 PMID:8471035

One Step Quantum Key Distribution Based on EPR Entanglement

PubMed Central

Li, Jian; Li, Na; Li, Lei-Lei; Wang, Tao

2016-01-01

A novel quantum key distribution protocol is presented, based on entanglement and dense coding and allowing asymptotically secure key distribution. Considering the storage time limit of quantum bits, a grouping quantum key distribution protocol is proposed, which overcomes the vulnerability of first protocol and improves the maneuverability. Moreover, a security analysis is given and a simple type of eavesdropper’s attack would introduce at least an error rate of 46.875%. Compared with the “Ping-pong” protocol involving two steps, the proposed protocol does not need to store the qubit and only involves one step. PMID:27357865

Purification, chemical modification and immunostimulating activity of polysaccharides from Tremella aurantialba fruit bodies*

PubMed Central

Du, Xiu-ju; Zhang, Jing-song; Yang, Yan; Tang, Qing-jiu; Jia, Wei; Pan, Ying-jie

2010-01-01

Ultrafiltration and a series of chromatographic steps were used to isolate and purify polysaccharides from Tremella aurantialba fruit bodies. Three crude fractions (TAP50w, TAP10–50w, and TAP1–10w), five semi-purified fractions (TAPA–TAPE), and one purified fraction (TAPA1) were obtained. A sulfated derivative of TAPA1 (TAPA1-s) was prepared by chemical modification. The immunostimulating activity of the polysaccharide fractions in vitro was determined using the mouse spleen lymphocyte proliferation assay. Of the three crude fractions tested, cell proliferation rates were increased most by TAP50w. Furthermore, TAPA1-s was markedly more stimulatory than TAPA1, indicating that sulfonation was an effective way to enhance the immunostimulating activity of polysaccharide. PMID:20506575

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.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Evaluation of a continuous-rotation, high-speed scanning protocol for micro-computed tomography.

PubMed

Kerl, Hans Ulrich; Isaza, Cristina T; Boll, Hanne; Schambach, Sebastian J; Nolte, Ingo S; Groden, Christoph; Brockmann, Marc A

2011-01-01

Micro-computed tomography is used frequently in preclinical in vivo research. Limiting factors are radiation dose and long scan times. The purpose of the study was to compare a standard step-and-shoot to a continuous-rotation, high-speed scanning protocol. Micro-computed tomography of a lead grid phantom and a rat femur was performed using a step-and-shoot and a continuous-rotation protocol. Detail discriminability and image quality were assessed by 3 radiologists. The signal-to-noise ratio and the modulation transfer function were calculated, and volumetric analyses of the femur were performed. The radiation dose of the scan protocols was measured using thermoluminescence dosimeters. The 40-second continuous-rotation protocol allowed a detail discriminability comparable to the step-and-shoot protocol at significantly lower radiation doses. No marked differences in volumetric or qualitative analyses were observed. Continuous-rotation micro-computed tomography significantly reduces scanning time and radiation dose without relevantly reducing image quality compared with a normal step-and-shoot protocol.

Development of a cross-disciplinary continuous insulin infusion protocol for non-critically ill patients in a French university hospital.

PubMed

Bernard, Lise; Roche, Béatrice; Batisse, Marie; Maqdasy, Salwan; Terral, Daniel; Sautou, Valérie; Tauveron, Igor

2016-10-01

In non-critically ill patients, the use of an insulin syringe pump allows the management of temporary situations during which other therapies cannot be used (failure of subcutaneous injections, awaiting advice from the diabetes team, emergency situations, prolonged corticosteroid therapy, initiation of an artificial nutrition, need for a fasting status, etc.). To manage the risks related to this «never event», the use of a standard validated protocol for insulin administration and monitoring is an essential prerequisite. To this end, a multidisciplinary approach is recommended. With the support of our subcommission «Endocrinology-Diabetology», we proceeded with a «step-by-step process» to create such a standardized protocol: (1) review of all existing protocols in our hospital; (2) overview of the literature data concerning insulin infusion protocols developed by multidisciplinary teams in France and abroad; (3) development of a standardized protocol for non-intensive care unit patients, respecting the current recommendations and adapting it to the working habits of health teams; and (4) validation of the protocol Two protocols based on the same structure but adapted to the health status of the patient have been developed. The protocols are divided in to three parts: (1) golden rules to make the use of the protocol appropriate and safe; (2) the algorithm (a double entry table) corresponding to a dynamic adaptation of insulin doses, clearly defining the target and the 'at risk situations'; and (3) practical aspects of the protocol: preparation of the syringe, treatment initiation and traceability. The protocols have been validated by the institution. Our standardized insulin infusion protocol is simple, easy to implement, safe and is likely to be applicable in diverse care units. However, the efficiency, safety and the workability of our protocols have to be clinically evaluated. © 2016 John Wiley & Sons, Ltd.

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. PMID:27105777

A dual protease approach for expression and affinity purification of recombinant proteins.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

PubMed

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Circular Dichroism Spectroscopy: Enhancing a Traditional Undergraduate Biochemistry Laboratory Experience

ERIC Educational Resources Information Center

Lewis, Russell L.; Seal, Erin L.; Lorts, Aimee R.; Stewart, Amanda L.

2017-01-01

The undergraduate biochemistry laboratory curriculum is designed to provide students with experience in protein isolation and purification protocols as well as various data analysis techniques, which enhance the biochemistry lecture course and give students a broad range of tools upon which to build in graduate level laboratories or once they…

Plaque assay for African swine fever virus on swine macrophages.

PubMed

Bustos, M J; Nogal, M L; Revilla, Y; Carrascosa, A L

2002-07-01

A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses.

Purification and Analysis of Colorful Hypothetical Open Reading Frames: An Inexpensive Gateway Laboratory

ERIC Educational Resources Information Center

DeSantis, Kara A.; Reinking, Jeffrey L.

2011-01-01

This laboratory exercise is an inquiry-based investigation developed around the core experiment where students, working alone or in groups, each purify and analyze their own prescreened colored proteins using immobilized metal affinity chromatography (IMAC). Here, we present reagents and protocols that allow 12 different proteins to be purified in…

Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

PubMed

Rodrigues, Simone M; Soares, Virgínia L F; de Oliveira, Tahise M; Gesteira, Abelmon S; Otoni, Wagner C; Costa, Marcio G C

2007-11-01

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

PubMed

Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

2016-03-01

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

ANALYSIS OF RICIN TOXIN PREPARATIONS FOR CARBOHYDRATE AND FATTY ACID ABUNDANCE AND ISOTOPE RATIO INFORMATION

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

Wunschel, David S.; Kreuzer-Martin, Helen W.; Antolick, Kathryn C.

2009-12-01

This report describes method development and preliminary evaluation for analyzing castor samples for signatures of purifying ricin. Ricin purification from the source castor seeds is essentially a problem of protein purification using common biochemical methods. Indications of protein purification will likely manifest themselves as removal of the non-protein fractions of the seed. Two major, non-protein, types of biochemical constituents in the seed are the castor oil and various carbohydrates. The oil comprises roughly half the seed weight while the carbohydrate component comprises roughly half of the remaining “mash” left after oil and hull removal. Different castor oil and carbohydrate componentsmore » can serve as indicators of specific toxin processing steps. Ricinoleic acid is a relatively unique fatty acid in nature and is the most abundant component of castor oil. The loss of ricinoleic acid indicates a step to remove oil from the seeds. The relative amounts of carbohydrates and carbohydrate-like compounds, including arabinose, xylose, myo-inositol fucose, rhamnose, glucosamine and mannose detected in the sample can also indicate specific processing steps. For instance, the differential loss of arabinose relative to mannose and N-acetyl glucosamine indicates enrichment for the protein fraction of the seed using protein precipitation. The methods developed in this project center on fatty acid and carbohydrate extraction from castor samples followed by derivatization to permit analysis by gas chromatography-mass spectrometry (GC-MS). Method descriptions herein include: the source and preparation of castor materials used for method evaluation, the equipment and description of procedure required for chemical derivatization, and the instrument parameters used in the analysis. Two types of derivatization methods describe analysis of carbohydrates and one procedure for analysis of fatty acids. Two types of GC-MS analysis is included in the method development, one employing a quadrupole MS system for compound identification and an isotope ratio MS for measuring the stable isotope ratios of deuterium and hydrogen (D/H) in fatty acids. Finally, the method for analyzing the compound abundance data is included. This study indicates that removal of ricinoleic acid is a conserved consequence of each processing step we tested. Furthermore, the stable isotope D/H ratio of ricinoleic acid distinguished between two of the three castor seed sources. Concentrations of arabinose, xylose, mannose, glucosamine and myo-inositol differentiated between crude or acetone extracted samples and samples produced by protein precipitation. Taken together these data illustrate the ability to distinguish between processes used to purify a ricin sample as well as potentially the source seeds.« less

Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius).

PubMed

Elamin, Babiker A; Al-Maleki, Abdulmajeed; Ismael, Mohammad A; Ayoub, Mohammed Akli

2014-12-01

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS-PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.

Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

PubMed Central

Elamin, Babiker A.; Al-Maleki, Abdulmajeed; Ismael, Mohammad A.; Ayoub, Mohammed Akli

2014-01-01

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries. PMID:25473366

M13 bacteriophage purification using poly(ionic liquids) as alternative separation matrices.

PubMed

Jacinto, Maria João; Patinha, David J S; Marrucho, Isabel M; Gonçalves, João; Willson, Richard C; Azevedo, Ana M; Aires-Barros, M Raquel

2018-01-12

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology. Copyright © 2017 Elsevier B.V. All rights reserved.

Simplified and robust one-step radiosynthesis of [18 F]DCFPyL via direct radiofluorination and cartridge-based purification.

PubMed

Dornan, Mark H; Simard, José-Mathieu; Leblond, Antoine; Juneau, Daniel; Delouya, Guila; Saad, Fred; Ménard, Cynthia; DaSilva, Jean N

2018-05-02

[ 18 F]DCFPyL is a clinical-stage PET radiotracer used to image prostate cancer. This report details the efficient production of [ 18 F]DCFPyL using single-step direct radiofluorination, without the use of carboxylic acid-protecting groups. Radiolabeling reaction optimization studies revealed an inverse correlation between the amount of precursor used and the radiochemical yield. This simplified approach enabled automated preparation of [ 18 F]DCFPyL within 28 minutes using HPLC purification (26% ± 6%, at EOS, n = 4), which was then scaled up for large-batch production to generate 1.46 ± 0.23 Ci of [ 18 F]DCFPyL at EOS (n = 7) in high molar activity (37 933 ± 4158 mCi/μmol, 1403 ± 153 GBq/μmol, at EOS, n = 7). Further, this work enabled the development of [ 18 F]DCFPyL production in 21 minutes using an easy cartridge-based purification (25% ± 9% radiochemical yield, at EOS, n = 3). Copyright © 2018 John Wiley & Sons, Ltd.

A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

ERIC Educational Resources Information Center

Farrell, Shawn O.; Choo, Darryl

1989-01-01

Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

PubMed

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

PubMed Central

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

PubMed Central

d'Azzo, Alessandra

2013-01-01

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein–protein interactions4,15. PMID:23486347

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

PubMed

Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

2011-08-01

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. Copyright © 2011 Elsevier B.V. All rights reserved.

MollDE: a homology modeling framework you can click with.

PubMed

Canutescu, Adrian A; Dunbrack, Roland L

2005-06-15

Molecular Integrated Development Environment (MolIDE) is an integrated application designed to provide homology modeling tools and protocols under a uniform, user-friendly graphical interface. Its main purpose is to combine the most frequent modeling steps in a semi-automatic, interactive way, guiding the user from the target protein sequence to the final three-dimensional protein structure. The typical basic homology modeling process is composed of building sequence profiles of the target sequence family, secondary structure prediction, sequence alignment with PDB structures, assisted alignment editing, side-chain prediction and loop building. All of these steps are available through a graphical user interface. MolIDE's user-friendly and streamlined interactive modeling protocol allows the user to focus on the important modeling questions, hiding from the user the raw data generation and conversion steps. MolIDE was designed from the ground up as an open-source, cross-platform, extensible framework. This allows developers to integrate additional third-party programs to MolIDE. http://dunbrack.fccc.edu/molide/molide.php rl_dunbrack@fccc.edu.

Highly efficient one-pot/one-step synthesis of multiblock copolymers from three-component polymerization of carbon dioxide, epoxide and lactone.

PubMed

Li, Yang; Hong, Jiali; Wei, Renjian; Zhang, Yingying; Tong, Zaizai; Zhang, Xinghong; Du, Binyang; Xu, Junting; Fan, Zhiqiang

2015-02-01

It is a long-standing challenge to combine mixed monomers into multiblock copolymer (MBC) in a one-pot/one-step polymerization manner. We report the first example of MBC with biodegradable polycarbonate and polyester blocks that were synthesized from highly efficient one-pot/one-step polymerization of cyclohexene oxide (CHO), CO 2 and ε-caprolactone (ε-CL) in the presence of zinc-cobalt double metal cyanide complex and stannous octoate. In this protocol, two cross-chain exchange reactions (CCER) occurred at dual catalysts respectively and connected two independent chain propagation procedures ( i.e. , polycarbonate formation and polyester formation) simultaneously in a block-by-block manner, affording MBC without tapering structure. The multiblock structure of MBC was determined by the rate ratio of CCER to the two chain propagations and could be simply tuned by various kinetic factors. This protocol is also of significance due to partial utilization of renewable CO 2 and improved mechanical properties of the resultant MBC.

Template-DTW based on inertial signals: Preliminary results for step characterization.

PubMed

Mantilla, Juan; Oudre, Laurent; Barrois, Remi; Vienne, Alienor; Ricard, Damien

2017-07-01

In this paper, we present a method for the creation of a library of inertial signals based on Dynamic Time Warping (DTW) for step characterization, with preliminary results in control subjects and patients with neurological diseases. Subjects performed a protocol including a 10 m straight walking, then turn back and walking for additional 10 m. The library is constructed with inertial signals (acceleration and angular velocities recorded in three directions) aligned with the DTW. Templates in the library are obtained for a specific cohort and for the different walking phases of the protocol. They are compared to the signal of a single subject by calculating a Pearson correlation coefficient. The method has been tested on a database of 864 exercises, obtained from 71 healthy controls, 24 patients with Parkinson disease and 48 patients with Radiation Induced Leukoencephalopathy (RIL). Pearson correlation classification reports a precision of about 85% for step detection. For exercise characterization the sensitivity is about 57%, 56% and 82% for Parkinson, RIL and control subjects respectively.

The Amateur Scientist.

ERIC Educational Resources Information Center

Walker, Jearl

1986-01-01

Relates how Charles Martin Hall discovered the method of making pure aluminum metal in 1886. Retraces the events and steps that led to the process of aluminum purification. Includes diagrams of the apparatus used in the investigations. (ML)

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography.

PubMed

Saavedra, Soledad L; Martínez Ceron, María C; Giudicessi, Silvana L; Forno, Guillermina; Bosco, María Belén; Marani, Mariela M; Erra-Balsells, Rosa; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

2018-04-25

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Clomiphene Stair-Step Protocol for Women With Polycystic Ovary Syndrome.

PubMed

Jones, Tiffanny; Ho, Jacqueline R; Gualtieri, Marc; Bruno-Gaston, Janet; Chung, Karine; Paulson, Richard J; Bendikson, Kristin A

2018-01-01

To compare time to ovulation, ovulation rates, and side effect profile of traditional and the stair-step protocol for ovulation induction using clomiphene citrate in women with polycystic ovary syndrome (PCOS). We performed a retrospective study of women seeking care for infertility with a diagnosis of PCOS at a university-based infertility clinic from July 2012 to July 2014. We included patients who were resistant to the initial starting dose of 50 mg clomiphene. The primary outcome was time to ovulation. Secondary outcomes included ovulation rates, clinical pregnancy rates, and mild and moderate-to-severe side effects based on dose. For the traditional protocol, higher doses of clomiphene were used each subsequent month if no ovulation occurred. For the stair-step protocol, higher doses of clomiphene were given 7 days after the last dose if no dominant follicles were seen on ultrasonography. Our study had 80% power to detect a 20% difference in ovulation. One hundred nine patients were included in the analysis with 66 (60.6%) in the traditional and 43 (39.4%) in the stair-step protocol. Age and body mass index were similar between groups. The time to ovulation was decreased in the stair-step protocol group compared with the traditional protocol group (23.1±0.9 days vs 47.5±6.3 days). Ovulation rates were increased in the stair-step group compared with the traditional group at 150 mg (16 [37%] vs 8 [12%], P=.004) and at 200 mg (9 [21%] vs 3 [5%], P=.01). Pregnancy rates were similar between groups once ovulation was achieved (12 [18.1%] vs 7 [16.3%], P=.08). The stair-step protocol had an increased incidence of mild side effects (vasomotor flushes, headaches, gastrointestinal disturbance, mastalgia, changes in mood; 18 [41%] vs 8 [12%]), but there was no difference in the incidence of severe side effects (headaches, visual disturbances). For women with PCOS, the stair-step clomiphene protocol is associated with decreased time to ovulation and increased ovulation rates at higher doses when compared with the traditional protocol.

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool

PubMed Central

Iwasaki, Yuko; Sugita, Sunao; Mandai, Michiko; Yonemura, Shigenobu; Onishi, Akishi; Ito, Shin-ichiro; Mochizuki, Manabu; Ohno-Matsui, Kyoko; Takahashi, Masayo

2016-01-01

Purpose To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC). Methods Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated. Results We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation. Conclusion We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE. PMID:27385038

Environmentally Friendly Procedure Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking for the Discovery of Potent Antiviral Compounds from Euphorbia semiperfoliata.

PubMed

Nothias, Louis-Félix; Boutet-Mercey, Stéphanie; Cachet, Xavier; De La Torre, Erick; Laboureur, Laurent; Gallard, Jean-François; Retailleau, Pascal; Brunelle, Alain; Dorrestein, Pieter C; Costa, Jean; Bedoya, Luis M; Roussi, Fanny; Leyssen, Pieter; Alcami, José; Paolini, Julien; Litaudon, Marc; Touboul, David

2017-10-27

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC 50 = 0.45 μM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC 50 = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

PubMed

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

[Bilateral labio-maxillo-palatal clefts. Therapeutic evaluation].

PubMed

Raphaël, B; Morand, B; Bettega, G; Lesne, V; Lesne, C; Lebeau, J

2001-06-01

The wide diversity of bilateral facial clefts makes it most difficult to assess surgical success, particularly in terms of long-term outcome. The aim of this work was to examine the rationale for the current protocol used for cleft surgery at the Grenoble University Hospital. In a first group of 28 children, a 3-step surgical protocol was applied. The first two steps were performed between 4 and 8 months with at least 3 months between each procedure. Skoog's unilateral cheilo-rhino-uranoplasty was used, associated with a periosteal tibial graft. The third step, performed between 10 and 12 months, was for staphylorraphy. Outcome was analyzed at 15 years and evidenced the deleterious effect of excessive and asymmetrical premaxillary scars, of the 2-step cheiloplasty and of columella lengthenings from the lip. The frequency of secondary revision of the superior labial vestibule and the medial labial tubercule (43%) was considered to be high; this procedure should be re-examined as should be osteotomy (32% revision). Palatine closure, acquired in 82% of the cases and premaxillary stability, achieved in 86%, would appear to favor use of the periosteal tibial graft. The osteogenic capacity of this graft tissue was less satisfactory after a second harvesting (from the same tibia three months later). These results have led us to modify our protocol, favoring early and total closure of the bony palate and continued use of the periosteal tibial graft. We now use the following operative protocol: premaxillary alignment using an active orthopedic plate at 2 months, lip adhesion associated with staphylorraphy and passive palatine contention plate at 3 months, definitive bilateral cheilo-uranoplasty associated with a single periosteal graft at 7 months. The preliminary results with this protocol in a group of 12 children have shown better quality scars, more harmonious maxillary arches, an excellent occlusion of the deciduous dentition, and preservation of the positive results obtained with the periosteal tibial graft.

Bioactivities, isolation and purification methods of polysaccharides from natural products: A review.

PubMed

Shi, Lei

2016-11-01

Polysaccharides play multiple roles and have extensive bioactivities in life process and an immense potential in healthcare, food and cosmetic industries, due to their therapeutic effects and relatively low toxicity. This review describes their major functions involved in antitumor, anti-virus, and anti-inflammatory bioactivities. Due to their enormous structural heterogeneity, the approaches for isolation and purification of polysaccharides are distinct from that of the other macromolecules such as proteins, etc. Yet, to achieve the homogeneity is the initial step for studies of polysaccharide structure, pharmacology, and its structure-activity relationships. According to the experiences accumulated by our lab and the published literatures, this review also introduces the methods widely used in isolation and purification of polysaccharides. Copyright © 2016 Elsevier B.V. All rights reserved.

Benchmarking a quantum teleportation protocol in superconducting circuits using tomography and an entanglement witness.

PubMed

Baur, M; Fedorov, A; Steffen, L; Filipp, S; da Silva, M P; Wallraff, A

2012-01-27

Teleportation of a quantum state may be used for distributing entanglement between distant qubits in quantum communication and for quantum computation. Here we demonstrate the implementation of a teleportation protocol, up to the single-shot measurement step, with superconducting qubits coupled to a microwave resonator. Using full quantum state tomography and evaluating an entanglement witness, we show that the protocol generates a genuine tripartite entangled state of all three qubits. Calculating the projection of the measured density matrix onto the basis states of two qubits allows us to reconstruct the teleported state. Repeating this procedure for a complete set of input states we find an average output state fidelity of 86%.

Crystallization of Macromolecules

PubMed Central

Friedmann, David; Messick, Troy; Marmorstein, Ronen

2014-01-01

X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

Slurried solid media for simultaneous water purification and carbon dioxide removal from gas mixtures

DOEpatents

Aines, Roger D.; Bourcier, William L.; Viani, Brian

2013-01-29

A slurried solid media for simultaneous water purification and carbon dioxide removal from gas mixtures includes the steps of dissolving the gas mixture and carbon dioxide in water providing a gas, carbon dioxide, water mixture; adding a porous solid media to the gas, carbon dioxide, water mixture forming a slurry of gas, carbon dioxide, water, and porous solid media; heating the slurry of gas, carbon dioxide, water, and porous solid media producing steam; and cooling the steam to produce purified water and carbon dioxide.