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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28088451/efficient-sortase-mediated-n-terminal-labeling-of-tev-protease-cleaved-recombinant-proteins
#1
Kwabena Sarpong, Ron Bose
A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein...
January 11, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28087367/production-of-recombinant-proteins-in-escherichia-coli-tagged-with-the-fusion-protein-cusf3h
#2
Teresa Vargas-Cortez, Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria, Xristo Zarate
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E...
January 10, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28056928/new-ligation-independent-cloning-vectors-for-expression-of-recombinant-proteins-with-a-self-cleaving-cpd-6xhis-tag
#3
Marco Biancucci, Jazel S Dolores, Jennifer Wong, Sarah Grimshaw, Wayne F Anderson, Karla J F Satchell, Keehwan Kwon
BACKGROUND: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. RESULTS: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag"...
January 5, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28054967/expression-purification-and-biophysical-characterization-of-a-secreted-anthrax-decoy-fusion-protein-in-nicotiana-benthamiana
#4
Kalimuthu Karuppanan, Sifti Duhra-Gill, Muchena J Kailemia, My L Phu, Carlito B Lebrilla, Abhaya M Dandekar, Raymond L Rodriguez, Somen Nandi, Karen A McDonald
Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation...
January 4, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28043587/cloning-expression-and-purification-of-recombinant-protein-mpt-64-from-a-virulent-strain-of-mycobacterium-bovis-in-a-prokaryotic-system
#5
Maryam Mohammadi Tashakkori, Majid Tebianian, Mohammad Tabatabaei, Nader Mosavari
OBJECTIVE: Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2...
December 2016: International Journal of Mycobacteriology
https://www.readbyqxmd.com/read/28042093/expression-and-antigenicity-of-recombinant-human-respiratory-syncytial-virus-glycoproteins-having-different-affinity-tags
#6
Han Saem Lee, A-Reum Kim, Kisoon Kim, Wan-Ji Lee, Sung Soon Kim, You-Jin Kim
Human respiratory syncytial virus (HRSV) is a main cause of lower respiratory tract infections in infants and the elderly. Glycoprotein (G) is major antigen on the viral surface, and plays a key role for virus entry. Therefore, purification of the glycoprotein of HRSV is critical for the development of HRSV vaccine and serological diagnosis. In this study, we report the design and characterization of glycoprotein engineered rationally to enhance the protein solubility and to facilitate efficient purification...
December 29, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/28025687/characterisation-of-the%C3%A2-daacs%C3%A2-family-escherichia-coli-glutamate-aspartate-proton-symporter-gltp-using-computational-chemical-biochemical-and-biophysical-methods
#7
Moazur Rahman, Fouzia Ismat, Li Jiao, Jocelyn M Baldwin, David J Sharples, Stephen A Baldwin, Simon G Patching
Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh...
December 26, 2016: Journal of Membrane Biology
https://www.readbyqxmd.com/read/28025125/production-purification-and-immunogenicity-of-recombinant-ebola-virus-proteins-a-comparison-of-freund-s-adjuvant-and-adjuvant-system-03
#8
Krister Melén, Laura Kakkola, Felix He, Kari Airenne, Olli Vapalahti, Helen Karlberg, Ali Mirazimi, Ilkka Julkunen
There is an urgent need for Ebola virus (EBOV) proteins, EBOV-specific antibodies and recombinant antigens to be used in diagnostics and as potential vaccine candidates. Our objective was to produce and purify recombinant proteins for immunological assays and for the production of polyclonal EBOV specific antibodies. In addition, a limited comparison of the adjuvant effects of Freund's complete adjuvant (FCA) and adjuvant system 03 (AS03) was carried out. Recombinant EBOV GST-VP24, -VP30, -VP35, -VP40 and -NP were produced in E...
December 23, 2016: Journal of Virological Methods
https://www.readbyqxmd.com/read/27989736/construction-of-pduo-a-bicistronic-shuttle-vector-series-for-dual-expression-of-recombinant-proteins
#9
Paul A Nakata
Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new bicistronic shuttle vector series, pDUO, for the dual expression of genes in different hosts. Each vector was designed and constructed to contain two araC-pBAD inducible promoter systems for tight control over gene expression...
December 16, 2016: Plasmid
https://www.readbyqxmd.com/read/27933303/purification-of-baculovirus-vectors-using-heparin-affinity-chromatography
#10
Md Nasimuzzaman, Danielle Lynn, Johannes Cm van der Loo, Punam Malik
Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells...
2016: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/27920885/cloning-and-expression-of-soluble-recombinant-hiv-1-crf35-protease-hp-thioredoxin-fusion-protein
#11
Asaad Azarnezhad, Zohreh Sharifi, Rahmatollah Seyedabadi, Arshad Hosseini, Behrooz Johari, Mahsa Sobhani Fard
BACKGROUND: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. METHODS: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning...
October 2016: Avicenna Journal of Medical Biotechnology
https://www.readbyqxmd.com/read/27920826/expression-and-purification-of-truncated-diphtheria-toxin-dt386-in-escherichia-coli-an-attempt-for-production-of-a-new-vaccine-against-diphtheria
#12
Fatemeh Shafiee, Mohammad Rabbani, Mahdi Behdani, Ali Jahanian-Najafabadi
The aim of this study was to produce a recombinant protein consisting of the catalytic and translocation domains of diphtheria toxin for its later application as a vaccine candidate against Corynebacterium diphtheria. To achieve this goal, at first, the amino acid sequence of DT386 was used for prediction of T and B cell epitopes using on-line servers. The DT386 coding sequence was synthesized and subcloned into the NcoI and XhoI sites of pET28a plasmid and recombinant pET28a plasmid was used to transform Escherichia coli BL21 (DE3) host cells...
October 2016: Research in Pharmaceutical Sciences
https://www.readbyqxmd.com/read/27895115/shared-subunits-of-tetrahymena-telomerase-holoenzyme-and-replication-protein-a-have-different-functions-in-different-cellular-complexes
#13
Heather E Upton, Henry Chan, Juli Feigon, Kathleen Collins
In most eukaryotes, telomere maintenance relies on telomeric repeat synthesis by a reverse transcriptase named telomerase. To synthesize telomeric repeats, the catalytic subunit telomerase reverse transcriptase (TERT) uses the RNA subunit (TER) as a template. In the ciliate Tetrahymena thermophila, the telomerase holoenzyme consists of TER, TERT, and eight additional proteins, including the telomeric repeat single-stranded DNA-binding protein Teb1 and its heterotrimer partners Teb2 and Teb3. Teb1 is paralogous to the large subunit of the general single-stranded DNA binding heterotrimer replication protein A (RPA)...
January 6, 2017: Journal of Biological Chemistry
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
#14
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
#15
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...
January 20, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/27881117/novel-prokaryotic-expression-of-thioredoxin-fused-insulinoma-associated-protein-tyrosine-phosphatase-2-ia-2-its-characterization-and-immunodiagnostic-application
#16
Luciano Lucas Guerra, Natalia Inés Faccinetti, Aldana Trabucchi, Bruno David Rovitto, Adriana Victoria Sabljic, Edgardo Poskus, Ruben Francisco Iacono, Silvina Noemí Valdez
BACKGROUND: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A)...
November 24, 2016: BMC Biotechnology
https://www.readbyqxmd.com/read/27867058/application-of-strep-tactin-xt-for-affinity-purification-of-twin-strep-tagged-cb2-a-g-protein-coupled-cannabinoid-receptor
#17
Alexei Yeliseev, Lioudmila Zoubak, Thomas G M Schmidt
Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. Here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin...
November 17, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27864058/a-novel-alkaline-surfactant-stable-keratinase-with-superior-feather-degrading-potential-based-on-library-screening-strategy
#18
Chang Su, Jin-Song Gong, Rong-Xian Zhang, Li-Yan Tao, Wen-Fang Dou, Dan-Dan Zhang, Heng Li, Zhen-Ming Lu, Zheng-Hong Xu, Jin-Song Shi
A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55°C, respectively. It could remain stable at pH 5.0-12.0 and 40-60°C. Metal ions such as Ca(2+), Mn(2+), Ag(+), Na(+), Mg(2+), Li(+), Sn(2+) (1mM) displayed positive influence on keratinase, and particularly, Ca(2+) exhibited remarkable improvement effect by 2...
February 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/27863173/heterologous-expression-and-purification-of-active-l-asparaginase-i-of-saccharomyces-cerevisiae-in-escherichia-coli-host
#19
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/27854020/production-and-purification-of-antibodies-against-histone-modifications
#20
Benoit Guillemette, Ian Hammond-Martel, Hugo Wurtele, Alain Verreault
Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies...
2017: Methods in Molecular Biology
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