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Purification his tagged protein

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https://www.readbyqxmd.com/read/27894314/self-assembly-of-hexahistidine-tagged-tobacco-etch-virus-capsid-protein-into-microfilaments-that-induce-igg2-specific-response-against-a-soluble-porcine-reproductive-and-respiratory-syndrome-virus-chimeric-protein
#1
Carlos Alberto Manuel-Cabrera, Alba Adriana Vallejo-Cardona, Eduardo Padilla-Camberos, Rodolfo Hernández-Gutiérrez, Sara Elisa Herrera-Rodríguez, Abel Gutiérrez-Ortega
BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP)...
November 29, 2016: Virology Journal
https://www.readbyqxmd.com/read/27888121/in-situ-affinity-purification-of-his-tagged-protein-a-from-bacillus-megaterium-cultivation-using-recyclable-superparamagnetic-iron-oxide-nanoparticles
#2
Johannes Gädke, Lennart Kleinfeldt, Chris Schubert, Manfred Rohde, Rebekka Biedendieck, Georg Garnweitner, Rainer Krull
This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time,>85% of the product was adsorbed onto the particles...
November 22, 2016: Journal of Biotechnology
https://www.readbyqxmd.com/read/27880036/protein-phosphotyrosine-proteome-profiling-by-superbinder-sh2-domain-affinity-purification-mass-spectrometry-ssh2-ap-ms
#3
Jiefei Tong, Biyin Cao, Gregory D Martyn, Jonathan R Krieger, Paul Taylor, Bradley Yates, Sachdev S Sidhu, Shawn S C Li, Xinliang Mao, Michael F Moran
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...
November 23, 2016: Proteomics
https://www.readbyqxmd.com/read/27873257/bacterial-histidine-kinases-overexpression-purification-and-inhibitor-screen
#4
Mike Gajdiss, Michael Türck, Gabriele Bierbaum
Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27863173/heterologous-expression-and-purification-of-active-l-asparaginase-i-of-saccharomyces-cerevisiae-in-escherichia-coli-host
#5
João H P M Santos, Iris M Costa, João V D Molino, Mariana S M Leite, Marcela V Pimenta, João A P Coutinho, Adalberto Pessoa, Sónia P M Ventura, André M Lopes, Gisele Monteiro
L-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract...
November 14, 2016: Biotechnology Progress
https://www.readbyqxmd.com/read/27837492/expression-and-purification-of-n-terminally-his-tagged-recombinant-type-iii-secretion-proteins
#6
Travis D Alvine, Patrick Osei-Owusu, Danielle L Jessen Condry, Matthew L Nilles
The ability to express and purify recombinant needle proteins from the Type III Secretion System (T3SS) of many gram-negative bacteria has allowed us to develop novel experimental approaches, both in vitro and in vivo, to identify unique roles for T3SS in bacterial pathogenesis. In addition, these purified needle proteins have shown to be promising immunotherapies acting as both protective antigens and adjuvants, presumably due to their immune activating properties. Here, we describe the expression and purification of recombinant T3SS needle proteins...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27815133/simple-purification-method-for-a-recombinantly-expressed-native-his-tag-free-aminopeptidase-a-from-lactobacillus-delbrueckii
#7
Timo Stressler, Coralie Tanzer, Jacob Ewert, Wolfgang Claaßen, Lutz Fischer
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 His6-tag for easier purification...
November 1, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27795879/gene-cloning-expression-and-polyclonal-antibody-preparation-of-rab3a-for-protein-interaction-analysis
#8
Xia Tang, Jia Chen, Ying Wang, Xianchun Wang
BACKGROUND: Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear. RESULTS: In this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step...
2016: SpringerPlus
https://www.readbyqxmd.com/read/27775592/prokaryotic-expression-purification-and-immunogenicity-in-rabbits-of-the-small-antigen-of-hepatitis-delta-virus
#9
Vera L Tunitskaya, Olesja V Eliseeva, Vladimir T Valuev-Elliston, Daria A Tyurina, Natalia F Zakirova, Olga A Khomich, Martins Kalis, Oleg E Latyshev, Elizaveta S Starodubova, Olga N Ivanova, Sergey N Kochetkov, Maria G Isaguliants, Alexander V Ivanov
Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits...
October 20, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27774953/-preparation-and-identification-of-rabbit-anti-akr1b10-polyclonal-antibody
#10
Jian Hu, Qingmei Wang, Li Huang, Yuanqing Zeng, Zheng Hu, Dixian Luo
Objective To prepare rabbit anti-aldo-keto reductase family 1 member B10 (AKR1B10) polyclonal antibody and identify its specificity. Methods AKR1B10 cDNA was amplified by reverse transcription PCR (RT-PCR) and inserted into prokaryotic expression vector pET-15b to form recombinant plasmid pET-15b-AKR1B10. The recombinant plasmid pET-15b-AKR1B10 was transformed into E.coli DH5α. Isopropylthio-β-D-galactoside (IPTG) was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/27773766/affinity-purification-of-bacterial-outer-membrane-vesicles-omvs-utilizing-a-his-tag-mutant
#11
Nathan J Alves, Kendrick B Turner, Kyle A DiVito, Michael A Daniele, Scott A Walper
To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin...
October 20, 2016: Research in Microbiology
https://www.readbyqxmd.com/read/27766259/high-efficient-expression-purification-and-functional-characterization-of-native-human-epidermal-growth-factor-in-escherichia-coli
#12
Yi Ma, Jieying Yu, Jinglian Lin, Shaomin Wu, Shan Li, Jufang Wang
Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10...
2016: BioMed Research International
https://www.readbyqxmd.com/read/27734368/production-of-recombinant-ccn-proteins-by-brevibacillus-choshinensis
#13
Hiroshi Hanagata, Makoto Mizukami
Brevibacillus choshinensis is an excellent host for the production of secretory proteins. This host has also been applied successfully to efficient production of CCN proteins. Described herein are methods of constructing plasmids for CCN protein production (IGFBP-, VWC-, TSP-, and CT-domain) with Brevibacillus as a host, cultivation methods for protein production, and methods of purification for domain proteins using his-tag.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27730558/purification-of-polyhistidine-tagged-proteins
#14
Sinéad T Loughran, Ronan T Bree, Dermot Walls
His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by Immobilized Metal Affinity Chromatography (IMAC). Here, we describe efficient procedures for the isolation of highly purified His-tagged target proteins from an E...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27730554/differential-precipitation-and-solubilization-of-proteins
#15
Barry J Ryan, Gemma K Kinsella
Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27721079/a-non-cleavable-hexahistidine-affinity-tag-at-the-carboxyl-terminus-of-the-hiv-1-pr55-gag-polyprotein-alters-nucleic-acid-binding-properties
#16
Maria C Bewley, Lisa Reinhart, Matthew S Stake, Shorena Nadaraia-Hoke, Leslie J Parent, John M Flanagan
HIV Gag (Pr55(Gag)), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55(Gag) has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6). In vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT (Endosomal Sorting Complexes Required for Transport) pathway, it has been difficult to study its role in the context of Gag using in vitro approaches...
October 6, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27704471/expression-purification-and-immobilization-of-tannase-from-staphylococcus-lugdunensis-mtcc-3614
#17
Amballa Chaitanyakumar, M Anbalagan
Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry...
December 2016: AMB Express
https://www.readbyqxmd.com/read/27672668/the-generation-and-characterization-of-recombinant-protein-and-antibodies-of-clostridium-perfringens-beta2-toxin
#18
Jin Zeng, Fuyang Song, Yi Yang, Chenjie Ma, Guangcun Deng, Yong Li, Yujiong Wang, Xiaoming Liu
Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence...
2016: Journal of Immunology Research
https://www.readbyqxmd.com/read/27664437/recombinant-production-purification-and-characterization-of-vessel-dilator-in-e-%C3%A2-coli
#19
Mahdi Abbasian, Hadieh Alsadat Eslampanah Seyedi, Badraldin Ebrahim Sayed Tabatabaei, Zahra Arab-Bafrani, Mohammad Reza Mofid, Reza Zareie
Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E...
January 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/27663563/expression-and-purification-of-tau-protein-and-its-frontotemporal-dementia-variants-using-a-cleavable-histidine-tag
#20
Thomas K Karikari, Alexandra Turner, Robert Stass, Leonie C Y Lee, Bethany Wilson, David A Nagel, Eric J Hill, Kevin G Moffat
Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods...
September 20, 2016: Protein Expression and Purification
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