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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28413818/data-on-enhanced-expression-and-purification-of-camelid-single-domain-antibodies-from-escherichia-coli-classical-inclusion-bodies
#1
Maristella Maggi, Claudia Scotti
Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells...
June 2017: Data in Brief
https://www.readbyqxmd.com/read/28409471/study-of-peroxisomal-protein-phosphorylation-by-functional-proteomics
#2
Andreas Schummer, Sven Fischer, Silke Oeljeklaus, Bettina Warscheid
Reversible protein phosphorylation is a frequently occurring posttranslational modification mediated by protein kinases and phosphatases that plays an essential role in the regulation of a large number of cellular processes. Evidence is accumulating that protein phosphorylation is also an important mechanism governing processes associated with peroxisome biology. For an improved and detailed understanding of these processes and their regulation it is therefore crucial to study phosphorylation of peroxisome-associated proteins and to determine the phosphorylated amino acid(s)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28386167/efficient-purification-protocol-for-bioengineering-allophycocyanin-trimer-with-n-terminus-histag
#3
Wenjun Li, Yang Pu, Na Gao, Zhihong Tang, Lufei Song, Song Qin
Allophycocyanin plays a key role for the photon energy transfer from the phycobilisome to reaction center chlorophylls with high efficiency in cyanobacteria. Previously, the high soluble self-assembled bioengineering allophycocyanin trimer with N-terminus polyhistidine from Synechocystis sp. PCC 6803 had been successfully recombined and expressed in Escherichia coli strain. The standard protocol with immobilized metal-ion affinity chromatography with chelating transition metal ion (Ni(2+)) was used to purify the recombinant protein...
March 2017: Saudi Journal of Biological Sciences
https://www.readbyqxmd.com/read/28381572/improving-the-expression-and-purification-of-soluble-recombinant-native-like-hiv-1-envelope-glycoprotein-trimers-by-targeted-sequence-changes
#4
Rajesh P Ringe, Gabriel Ozorowski, Anila Yasmeen, Albert Cupo, Victor M Cruz Portillo, Pavel Pugach, Michael Golabek, Kimmo Rantalainen, Lauren G Holden, Christopher A Cottrell, Ian A Wilson, Rogier W Sanders, Andrew B Ward, P J Klasse, John P Moore
Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (Tier-2) clade C virus. As appropriately purified, native-like CZA97...
April 5, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28349680/expression-and-purification-of-zasp-subdomains-and-clinically-important-isoforms-high-affinity-binding-to-g-actin
#5
Norman R Watts, Xiaolei Zhuang, Joshua D Kaufman, Ira W Palmer, Altaira D Dearborn, Stephen Coscia, Yotam Blech-Hermoni, Caterina Alfano, Annalisa Pastore, Ami Mankodi, Paul T Wingfield
Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown...
April 3, 2017: Biochemistry
https://www.readbyqxmd.com/read/28342173/metal-ions-binding-t4-lysozyme-as-an-intramolecular-protein-purification-tag-compatible-with-x-ray-crystallography
#6
Evzen Boura, Adriana Baumlova, Dominika Chalupska, Anna Dubankova, Martin Klima
Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag...
March 25, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28330196/cloning-and-expression-of-saccharomyces-cerevisiae-suc2-gene-in-yeast-platform-and-characterization-of-recombinant-enzyme-biochemical-properties
#7
Nooshin Mohandesi, Seyed Omid Ranaei Siadat, Kamahldin Haghbeen, Ardeshir Hesampour
Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28326614/recombinant-expression-of-porcine-spermadhesin-awn-and-its-phospholipid-interaction-indication-for-a-novel-lipid-binding-property
#8
F Schröter, K Müller, P Müller, E Krause, B C Braun
AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity...
March 21, 2017: Reproduction in Domestic Animals, Zuchthygiene
https://www.readbyqxmd.com/read/28315745/production-of-low-expressing-recombinant-cationic-biopolymers-with-high-purity
#9
Xuguang Chen, Alireza Nomani, Niket Patel, Arash Hatefi
The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications...
March 16, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28300559/expression-purification-and-in-vitro-refolding-of-the-recombinant-truncated-saposin-like-protein-2-antigen-for-development-of-diagnosis-of-human-fascioliasis
#10
Abolfazl Mirzadeh, Zarrintaj Valadkhani, Asiyeh Yoosefy, Jalal Babaie, Majid Golkar, Ahmad Reza Esmaeili Rastaghi, Elham Kazemi-Rad, Keyhan Ashrafi
Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle(®) T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC)...
March 11, 2017: Acta Tropica
https://www.readbyqxmd.com/read/28299729/expression-and-purification-of-matrix-metalloproteinases-in-escherichia-coli
#11
Krishna K Singh, Ruchi Jain, Harini Ramanan, Deepak K Saini
The MMP (matrix metalloproteinases) family of endopeptidases are involved in cleavage induced remodelling of the extracellular matrix including collagen, fibrinogen, elastin, and gelatin. Owing to their proteolytic activity which can cleave and degrade multiple intracellular substrates, the overexpression and purification of these proteins tends to be toxic. Here we describe a novel "matrix assisted refolding" protocol to overcome the technical challenges associated with overexpression and purification of full-length MMPs...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28296951/in-silico-design-expression-and-purification-of-novel-chimeric-escherichia-coli-o157-h7-ompa-fused-to-ltb-protein-in-escherichia-coli
#12
Aytak Novinrooz, Taghi Zahraei Salehi, Roya Firouzi, Sina Arabshahi, Abdollah Derakhshandeh
E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E...
2017: PloS One
https://www.readbyqxmd.com/read/28294503/exploring-protein-structure-and-dynamics-through-a-project-oriented-biochemistry-laboratory-module
#13
James M Lipchock, Patrick S Ginther, Bonnie B Douglas, Kelly E Bird, J Patrick Loria
Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics...
March 13, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28274101/enhanced-purification-of-recombinant-rat-nadph-p450-reductase-by-using-a-hexahistidine-tag
#14
Hyoung-Goo Park, Young-Ran Lim, Songhee Han, Dabin Jeong, Donghak Kim
NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum (ER) membrane and utilizes FMN, FAD, and NADPH as cofactors. While NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP⁺ in the affinity chromatography process...
March 9, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28265494/purification-and-biochemical-characterization-of-recombinant-persicaria-minor%C3%A2-%C3%AE-sesquiphellandrene-synthase
#15
De-Sheng Ker, Sze Lei Pang, Noor Farhan Othman, Sekar Kumaran, Ee Fun Tan, Thiba Krishnan, Kok Gan Chan, Roohaida Othman, Maizom Hassan, Chyan Leong Ng
BACKGROUND: Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment...
2017: PeerJ
https://www.readbyqxmd.com/read/28249301/recombinant-human-acidic-fibroblast-growth-factor-afgf-expressed-in-nicotiana-benthamiana-potentially-inhibits-skin-photoaging
#16
Jang-Ho Ha, Ha-Neul Kim, Ki-Beom Moon, Jae-Heung Jeon, Dai-Hyun Jung, Su-Jung Kim, Hugh S Mason, Seo-Yeon Shin, Hyun-Soon Kim, Kyung-Mok Park
Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident...
March 1, 2017: Planta Medica
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
#17
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
#18
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
#19
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/glycosylation-benchmark-profile-for-hiv-1-envelope-glycoprotein-production-based-on-eleven-env-trimers
#20
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 (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals...
May 1, 2017: Journal of Virology
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