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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28220817/a-simple-approach-for-preparation-of-affinity-matrices-simultaneous-purification-and-reversible-immobilization-of-a-streptavidin-mutein-to-agarose-matrix
#1
Sau-Ching Wu, Chris Wang, Dave Hansen, Sui-Lam Wong
SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain...
February 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28216490/-expression-and-purification-of-foxo1-dna-binding-domain-and-its-dna-properties
#2
Yinuer Ha, Jun Li, Yongheng Chen, Lin Chen, Zhuchu Chen
To express and purify FOXO1 DNA binding domain (DBD) and to evaluate its DNA binding properties.
 Methods: Gene sequence of FOXO1-DBD was optimized and the recombinant strains were induced at low temperature to obtain soluble protein of FOXO1-DBD. Then, FOXO1-DBD protein was purified through Ni affinity chromatography and cation exchange. Finally, the DNA binding properties of FOXO1-DBD was evaluated by electrophoretic mobility shift assay (EMSA).
 Results: Most of soluble proteins were obtained by optimizing its genes sequence and induction at 21 ℃...
January 28, 2017: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/28215401/nickel-salen-supported-paramagnetic-nanoparticles-for-6-his-target-recombinant-protein-affinity-purification
#3
Zahra Rashid, Ramin Ghahremanzadeh, Mohammad-Reza Nejadmoghaddam, Mahboobeh Nazari, Mohammad-Reza Shokri, Hossein Naeimi, Amir-Hassan Zarnani
In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc)2...
February 13, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28202756/a-glycosylation-benchmark-profile-for-hiv-1-envelope-glycoprotein-production-based-on-eleven-env-trimers
#4
Eden P Go, Haitao Ding, Shijian Zhang, Rajesh P Ringe, Nathan Nicely, David Hua, Robert T Steinbock, Michael Golabek, James Alin, S Munir Alam, Albert Cupo, Barton F Haynes, John C Kappes, John P Moore, Joseph G Sodroski, Heather Desaire
HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals: 1) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs...
February 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28198449/recombinant-vacuolar-iron-transporter-family-homologue-pfvit-from-human-malaria-causing-plasmodium-falciparum-is-a-fe-2-h-exchanger
#5
Paola Labarbuta, Katie Duckett, Catherine H Botting, Osama Chahrour, John Malone, John P Dalton, Christopher J Law
Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe(2+) into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells...
February 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28193333/heterologous-expression-of-%C3%AE-glutamyl-transpeptidase-from-bacillus-atrophaeus-gs-16-and-its-application-in-the-synthesis-of-%C3%AE-d-glutamyl-l-tryptophan-a-known-immunomodulatory-peptide
#6
Meenu Saini, Shruti Bindal, Rani Gupta
Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5...
April 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28181316/an-improved-method-for-purification-and-refolding-of-recombinant-hiv-vif-expressed-in-e-coli
#7
Carlos A Duarte, Mickel Palomino
Virion infectivity factor (Vif) is a 23 KDa protein which protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the Elongin B and C subunits, Cullin 5 and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein...
February 8, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28177783/expression-purification-and-renaturation-of-recombinant-peptide-based-hiv-vaccine-in-escherichia-coli
#8
Wei He, Jiayi Shu, Jian Zhang, Zhihua Liu, Jianqing Xu, Xia Jin, Xuedong Wang
One of the new HIV vaccine strategies is to design epitope-based immunogen. We previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen (HLA) DR types. In order to test whether this vaccine is immunogenic in vivo, a corresponding antigen needs to be prepared. To this end, we constructed recombinant plasmid containing DNA encoding 300 amino acids of this vaccine protein, and then transformed Escherichia coli BL21 (DE3) cells with the plasmid...
January 27, 2017: Canadian Journal of Microbiology
https://www.readbyqxmd.com/read/28168572/expression-purification-and-functionality-of-bioactive-recombinant-human-vascular-endothelial-growth-factor-vegf165-in-e-coli
#9
Awatef Taktak-BenAmar, Maram Morjen, Hazem Ben Mabrouk, Rania Abdelmaksoud-Dammak, Mohamed Guerfali, Najla Fourati-Masmoudi, Naziha Marrakchi, Ali Gargouri
Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28164628/evaluation-of-truncated-hcv-ns3-protein-for-potential-applications-in-immunization-and-diagnosis
#10
Foozieh Javadi, Pooneh Rahimi, Mohammad Hossien Modarressi, Azam Bolhassani, Mehdi Shafiee Ardestani, Seyed Mehdi Sadat, Mohammad Reza Aghasadeghi
BACKGROUND: Hepatitis C virus infection is one of global health concern. No vaccine is available so far and nonstructural protein 3 (NS3) is one of the main target antigens for developing studies on therapeutic vaccine and diagnostic application. In the current study, we expressed a truncated recombinant HCV-NS3 protein under native condition and evaluated its potential applications in immunization and diagnosis. METHODS: The recombinant pET-NS3 containing a truncated form of HCV NS3 region was constructed, confirmed by sequencing reactions, and expressed into E...
July 1, 2016: Clinical Laboratory
https://www.readbyqxmd.com/read/28153778/accelerating-the-clinical-development-of-protein-based-vaccines-for-malaria-by-efficient-purification-using-a-four-amino-acid-c-terminal-c-tag
#11
Jing Jin, Kathryn A Hjerrild, Sarah E Silk, Rebecca E Brown, Geneviève M Labbé, Jennifer M Marshall, Katherine E Wright, Sandra Bezemer, Stine B Clemmensen, Sumi Biswas, Yuanyuan Li, Aadil El-Turabi, Alexander D Douglas, Pim Hermans, Frank J Detmers, Willem A de Jongh, Matthew K Higgins, Rebecca Ashfield, Simon J Draper
Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example...
January 30, 2017: International Journal for Parasitology
https://www.readbyqxmd.com/read/28137655/a-fully-automated-procedure-for-the-parallel-multidimensional-purification-and-nucleotide-loading-of-the-human-gtpases-kras-rac1-and-ralb
#12
Christopher H Gray, Jennifer Konczal, Mokdad Mezna, Shehab Ismail, Justin Bower, Martin Drysdale
Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form...
January 28, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28126552/design-and-expression-of-recombinant-toxins-from-mexican-scorpions-of-the-genus-centruroides-for-production-of-antivenoms
#13
J M Jiménez-Vargas, V Quintero-Hernández, L González-Morales, E Ortiz, L D Possani
This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3)...
January 23, 2017: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/28125612/a-recombinant-human-anti-platelet-scfv-antibody-produced-in-pichia-pastoris-for-atheroma-targeting
#14
Amelie Vallet-Courbin, Mélusine Larivière, Agnès Hocquellet, Audrey Hemadou, Sarjapura-Nagaraja Parimala, Jeanny Laroche-Traineau, Xavier Santarelli, Gisèle Clofent-Sanchez, Marie-Josée Jacobin-Valat, Abdelmajid Noubhani
Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets...
2017: PloS One
https://www.readbyqxmd.com/read/28115011/recombinant-production-of-the-antibody-fragment-d1-3-scfv-with-different-bacillus-strains
#15
Antonia Lakowitz, Rainer Krull, Rebekka Biedendieck
BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments...
January 23, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28112396/a-novel-approach-to-characterize-host-cell-proteins-associated-with-therapeutic-monoclonal-antibodies
#16
Andrew S Thomson, Shing Mai, Michael P Byrne
Recombinant monoclonal antibody (mAb) products are widely produced in the pharmaceutical industry using Chinese hamster ovary (CHO) cells. Host cell proteins (HCPs) are one of many process-related impurities generated during the production of mAb products. The multi-analyte HCP enzyme linked immunosorbent assay (ELISA) is the industry standard accepted assay to measure clearance of HCPs from recombinant protein therapeutics. While similar platform processes are used for expression and purification, varying amounts of HCPs are found in final drug substances for different mAb products...
January 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28105864/expression-and-purification-of-l-asparaginase-from-escherichia-coli-and-the-inhibitory-effects-of-cyclic-dipeptides
#17
Yanan Zhang, Dan Li, Yan Li
L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB...
January 20, 2017: Natural Product Research
https://www.readbyqxmd.com/read/28092033/purification-of-socs-suppressor-of-cytokine-signaling-sh2-domains-for-structural-and-functional-studies
#18
Nicholas P D Liau, Artem Laktyushin, Jeffrey J Babon
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...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092031/expression-and-production-of-sh2-domain-proteins
#19
Bernard A Liu, Mari Ogiue-Ikeda, Kazuya Machida
The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28089451/crystallogenesis-of-membrane-proteins-mediated-by-polymer-bounded-lipid-nanodiscs
#20
Jana Broecker, Bryan T Eger, Oliver P Ernst
For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization...
February 7, 2017: Structure
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