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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28642366/saccharomyces-cerevisiae-red1-protein-exhibits-nonhomologous-dna-end-joining-activity-and-potentiates-hop1-promoted-pairing-of-double-stranded-dna
#1
Rucha Kshirsagar, Indrajeet Ghodke, Kalappa Muniyappa
Elucidation of the function of synaptonemal complex (SC) in <Saccharomyces cerevisiae> has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of different SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-specific DNA-binding protein that exhibits high affinity for the Holliday junction, promotes DNA bridging, condensation and pairing between duplex DNA molecules...
June 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28640945/production-of-human-vitronectin-in-nicotiana-benthamiana-using-the-inpact-hyper-expression-platform
#2
Benjamin Dugdale, Maiko Kato, Pradeep Deo, Manuel Plan, Mark Harrison, Robyn Lloyd, Terry Walsh, Robert Harding, James Dale
Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum-free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant Activation (INPACT) hyper-expression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol-inducible AlcR:alcA gene switch...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28630382/immunodiagnostic-value-of-echinococcus-granulosus-recombinant-b8-1-subunit-of-antigen-b
#3
Amir Savardashtaki, Bahador Sarkari, Farzane Arianfar, Zohreh Mostafavi-Pour
BACKGROUND: Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. OBJECTIVE: The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. METHODS: The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized...
June 2017: Iranian Journal of Immunology: IJI
https://www.readbyqxmd.com/read/28625911/white-shrimp-litopenaeus-vannamei-recombinant-lactate-dehydrogenase-biochemical-and-kinetic-characterization
#4
Ambar A Fregoso-Peñuñuri, Elisa M Valenzuela-Soto, Ciria G Figueroa-Soto, Alma B Peregrino-Uriarte, Manuel Ochoa-Valdez, Lilia Leyva-Carrillo, Gloria Yepiz-Plascencia
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28615092/-purification-of-the-recombinant-com1-and-adaa-of-coxiella-burnetii-and-identification-of-the-antigenicity
#5
Jingxian Liu, Yihong Ji, Zhiyang Shi, Yongjun Jiao
Objective To express and purify two kinds of antigens of Coxiella burnetii (C. burnetii), the main outer membrane protein Com1 and the acute disease antigen A (adaA), in prokaryotic expression system and to validate the two recombinant antigens by mass spectrometry and identify their antigenicity. Methods The gene sequences encoding Com1 and adaA were separately synthesized and constructed into the prokaryotic expression vector pET-20b(+). The constructed vectors were transformed into E.coli BL21(DE3), and the recombinant proteins were induced by IPTG...
June 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28612560/-construction-expression-of-her%C3%AE-lbd-prokaryotic-vector-and-the-activity-of-expressed-protein
#6
Yan-Fang Wang, Ji-Jun Zhao, Zai-Min Tian, Yan-Feng Guo
OBJECTIVES: To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain (hERα-LBD) and to evaluate the estrogen receptor ligand binding activity of the expressed protein. METHODS: hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a...
January 2017: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
https://www.readbyqxmd.com/read/28573567/expression-and-purification-of-recombinant-proteins-in-escherichia-coli-with-a-his6-or-dual-his6-mbp-tag
#7
Sreejith Raran-Kurussi, David S Waugh
Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28564596/the-histone-variant-macroh2a1-is-a-brca1%C3%A2-ubiquitin-ligase-substrate
#8
Beom-Jun Kim, Doug W Chan, Sung Yun Jung, Yue Chen, Jun Qin, Yi Wang
The breast- and ovarian-cancer-specific tumor suppressor BRCA1 and its heterodimeric partner BARD1 contain RING domains that implicate them as E3 ubiquitin ligases. Despite extensive efforts, the bona fide substrates of BRCA1/BARD1 remain elusive. Here, we used recombinant GST fused to four UBA domains to enrich ubiquitinated proteins followed by a Lys-ε-Gly-Gly (diGly) antibody to enrich ubiquitinated tryptic peptides. This tandem affinity purification method coupled with mass spectrometry identified 101 putative BRCA1/BARD1 E3 substrates...
May 30, 2017: Cell Reports
https://www.readbyqxmd.com/read/28545467/expression-of-deleted-atoxic-atypical-recombinant-beta2-toxin-in-a-baculovirus-system-and-production-of-polyclonal-and-monoclonal-antibodies
#9
Anna Serroni, Chiara Francesca Magistrali, Giovanni Pezzotti, Luca Bano, Martina Pellegrini, Giulio Severi, Chiara Di Pancrazio, Mirella Luciani, Manuela Tittarelli, Silvia Tofani, Antonio De Giuseppe
BACKGROUND: Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen...
May 25, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28542437/cp5-system-for-simple-and-highly-efficient-protein-purification-with-a-c-terminal-designed-mini-tag
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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