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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28542437/cp5-system-for-simple-and-highly-efficient-protein-purification-with-a-c-terminal-designed-mini-tag
#1
Hiroyuki Takeda, Wei Zhou, Kohki Kido, Ryoji Suno, Takahiro Iwasaki, Takuya Kobayashi, Tatsuya Sawasaki
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence)...
2017: PloS One
https://www.readbyqxmd.com/read/28510356/purification-of-recombinant-a%C3%AE-1-42-and-pglu-a%C3%AE-3-42-using-preparative-sds-page
#2
Claudia Spahn, Michael Wermann, Rico Eichentopf, Gerd Hause, Dagmar Schlenzig, Stephan Schilling
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...
May 16, 2017: Electrophoresis
https://www.readbyqxmd.com/read/28500928/molecular-cloning-characterization-and-in-silico-analysis-of-a-thermostable-%C3%AE-glucosidase-enzyme-from-putranjiva-roxburghii-with-a-significant-activity-for-cellobiose
#3
Bibekananda Kar, Preeti Verma, Girijesh Kumar Patel, Ashwani Kumar Sharma
The native Putranjiva roxburghii family 1 glycoside hydrolase enzyme showed β-D-fucosidase activity in addition to β-D-glucosidase and β-D-galactosidase activities reported in our previous study. A single step concanvalin A affinity chromatography for native PRGH1 improved the yield and reduced the purification time. The PRGH1 gene was cloned and overexpressed in E. coli. The full length gene contained an ORF of 1617 bp encoding a polypeptide of 538 amino acids. The amino acid sequence of PRGH1 showed maximum similarities to β-glucosidases and myrosinases...
May 10, 2017: Phytochemistry
https://www.readbyqxmd.com/read/28496951/can-aptameric-ligands-specific-to-plasma-coagulation-factor-vii-bind-the-recombinant-form-with-high-affinity-affinity-measurement-by-fluorescence-method
#4
Maryam Tabarzad, Marzieh Jafari, Nastaran Nafissi-Varcheh
BACKGROUND: Among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven(™), has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products...
April 2017: Avicenna Journal of Medical Biotechnology
https://www.readbyqxmd.com/read/28490692/-identification-of-the-interacting-proteins-with-s100a8-or-s100a9-by-affinity-purification-and-mass-spectrometry
#5
Jing Wang, Xuemei Zhang, Zheng Li, Xiayu Li, Jian Ma, Shourong Shen
To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins...
April 28, 2017: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/28487257/functional-efficacy-of-human-recombinant-fgf-2s-tagged-with-his-6-and-his-asn-6-at-the-n-and-c-termini-in-human-gingival-fibroblast-and-periodontal-ligament-derived-cells
#6
Ji-Hye Lee, Ji-Eun Lee, Kyung-Jung Kang, Young-Joo Jang
Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein...
May 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28471227/from-in-silico-immunogenicity-verification-to-in-vitro-expression-of-recombinant-core-ns3-fusion-protein-of-hcv
#7
S Hekmat, S D Siadat, M R Aghasadeghi, S M Sadat, G Bahramali, M M Aslani, M Mahdavi, S Shahbazi
BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is a serious global health burden. There is no effective vaccine against HCV and new direct acting antivirals (DAAs) are so expensive and virtually unavailable to the public. Therefore, seeking for therapeutic or prophylactic vaccines is exigent and reliever. METHODS: The secondary and tertiary structures of the recombinant Core-NS3 (rC-N) fusion protein of HCV and its B and T-cells epitopes were evaluated with bioinformatics software...
2017: Bratislavské Lekárske Listy
https://www.readbyqxmd.com/read/28470610/production-of-protein-kinases-in-e-coli
#8
Charlotte A Dodson
Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28470608/removal-of-affinity-tags-with-tev-protease
#9
Sreejith Raran-Kurussi, Scott Cherry, Di Zhang, David S Waugh
Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. This chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (TEV) protease in Escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Further, we cleave a model substrate protein (MBP-NusG) in vitro using the purified TEV protease to illustrate a protease cleavage protocol that can be employed for simple pilot experiments and large-scale protein preparations...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28465181/expression-purification-and-characterization-of-recombinant-8%C3%A2-kda-gelsolin-fragment
#10
Qing Zhang, Weijie Lu, Lina Ji, Zi-Chun Hua
A mutation (D187N/Y) in human plasma gelsolin (GSN) leads to the generation of an 8 kDa GSN fragment (8 kDa-GSN), and consequently causes the familial amyloidosis of Finnish type. Because of its faster kinetics of amyloid formation under physiologically relevant conditions, 8 kDa-GSN is used to explore gelsolin amyloidosis and screen small molecules that can disaggregate amyloids. However, the synthetic 8 kDa-GSN is expensive, and substantial quantities of 8 kDa-GSN are needed for the screen. Here we report a study to obtain recombinant 8 kDa-GSN with high yield from Escherichia coli...
April 29, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28446197/re-directing-bacterial-microcompartment-systems-to-enhance-recombinant-expression-of-lysis-protein-e-from-bacteriophage-%C3%AF-x174-in-escherichia-coli
#11
Mimi C Yung, Feliza A Bourguet, Timothy S Carpenter, Matthew A Coleman
BACKGROUND: Recombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates...
April 26, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28432439/characterization-of-a-functional-recombinant-human-creatine-kinase-mb-isoenzyme-prepared-by-tandem-affinity-purification-from-escherichia-coli
#12
Lihui Zou, Wen Su, Meng Wang, Wei Huang, Haijian Zhao, Enyi Zhang, Junhua Jin, Hongtao Xu, Fei Xiao
Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories...
April 21, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28413818/data-on-enhanced-expression-and-purification-of-camelid-single-domain-antibodies-from-escherichia-coli-classical-inclusion-bodies
#13
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
#14
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
#15
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
#16
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
#17
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 11, 2017: Biochemistry
https://www.readbyqxmd.com/read/28342173/metal-ions-binding-t4-lysozyme-as-an-intramolecular-protein-purification-tag-compatible-with-x-ray-crystallography
#18
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...
June 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
#19
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
#20
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
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