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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/27933303/purification-of-baculovirus-vectors-using-heparin-affinity-chromatography
#1
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
#2
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
#3
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
#4
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)...
November 28, 2016: 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
#5
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
#6
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/27881117/novel-prokaryotic-expression-of-thioredoxin-fused-insulinoma-associated-protein-tyrosine-phosphatase-2-ia-2-its-characterization-and-immunodiagnostic-application
#7
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
#8
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
#9
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...
November 15, 2016: 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
#10
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
#11
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
https://www.readbyqxmd.com/read/27826720/optimization-of-the-fermentation-and-downstream-processes-for-human-enterokinase-production-in-pichia-pastoris
#12
Kristína Melicherová, Ján Krahulec, Martin Šafránek, Veronika Lišková, Diana Hopková, Diana Széliová, Ján Turňa
Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml...
November 8, 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27826019/molecular-cloning-expression-and-ige-immunoreactivity-of-phospholipase-a1-a-major-allergen-from-polybia-paulista-hymenoptera-vespidae-venom
#13
Amilcar Perez-Riverol, Franco Dani Campos Pereira, Alexis Musacchio Lasa, Luis Gustavo Romani Fernandes, José Roberto Aparecido Dos Santos-Pinto, Débora Lais Justo-Jacomini, Gabriel Oliveira de Azevedo, Murilo Luiz Bazon, Mario Sergio Palma, Ricardo de Lima Zollner, Márcia Regina Brochetto-Braga
Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P...
December 15, 2016: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/27797005/purification-of-antibacterial-chapk-protein-using-a-self-cleaving-fusion-tag-and-its-activity-against-methicillin-resistant-staphylococcus-aureus
#14
Elahe Seyed Hosseini, Rezvan Moniri, Yasaman Dasteh Goli, Hamed Haddad Kashani
Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification...
October 28, 2016: Probiotics and Antimicrobial Proteins
https://www.readbyqxmd.com/read/27795422/native-folding-of-a-recombinant-gpe1-gpe2-heterodimer-vaccine-antigen-from-a-precursor-protein-fused-with-fc-igg
#15
Michael Logan, John Law, Jason Alexander Ji-Xhin Wong, Darren Hockman, Amir Landi, Chao Chen, Kevin Crawford, Juthika Kundu, Lesley Baldwin, Janelle Johnson, Anita Dahiya, Gerald LaChance, Joseph Marcotrigiano, Mansun Law, Steven Foung, Lorne Tyrrell, Michael Houghton
: A recombinant HCV-1 (1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production...
October 19, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27795419/an-in-vitro-rna-synthesis-assay-for-rabies-virus-defines-critical-ribonucleoprotein-interactions-for-polymerase-activity
#16
Benjamin Morin, Bo Liang, Erica Gardner, Robin A Ross, Sean P J Whelan
: We report an in vitro RNA synthesis assay for the RNA dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19-nt of the rabies virus genome we demonstrate that L alone initiates synthesis on naked RNA, and that P serves to enhance initiation and processivity of the RdRP...
October 19, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27787841/expression-and-purification-of-class-7-semaphorin-and-its-plexinc1-receptor-using-baculovirus-mediated-mammalian-cell-gene-transduction
#17
Xiaoyan Chen, Po-Han Chen, Xiaolin He
Semaphorins and their receptor plexins are large glycoproteins that are difficult to express using regular recombinant methods, and the widely used E. coli and baculovirus-insect cell systems have been inadequate for semaphorins and plexins which contain a large number of domains and are heavily modified by glycosylation. Here, we describe the expression of class 7 semaphorin (Sema7A) and the extracellular domain of its receptors PlexinC1, using the baculovirus-mediated mammalian cell gene transduction (BacMam) method...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27775592/prokaryotic-expression-purification-and-immunogenicity-in-rabbits-of-the-small-antigen-of-hepatitis-delta-virus
#18
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/27773757/purification-enzymatic-activity-and-inhibitor-discovery-for-recombinant-human-carbonic-anhydrase-xiv
#19
Vaida Juozapaitienė, Brigita Bartkutė, Vilma Michailovienė, Audrius Zakšauskas, Lina Baranauskienė, Sandra Satkūnė, Daumantas Matulis
Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E...
October 20, 2016: Journal of Biotechnology
https://www.readbyqxmd.com/read/27773392/tryptophan-tags-and-de-novo-designed-complementary-affinity-ligands-for-the-expression-and-purification-of-recombinant-proteins
#20
Ana Sofia Pina, Sara Carvalho, Ana Margarida G C Dias, Márcia Guilherme, Alice S Pereira, Luciana T Caraça, Ana Sofia Coroadinha, Christopher R Lowe, A Cecília A Roque
A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml(-1) and 0.48mgml(-1) for WFWFWF and NWNWNW, respectively...
October 19, 2016: Journal of Chromatography. A
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