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Protein expression and purification

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https://www.readbyqxmd.com/read/27920885/cloning-and-expression-of-soluble-recombinant-hiv-1-crf35-protease-hp-thioredoxin-fusion-protein
#1
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
#2
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/27920276/studies-of-recombinant-twa1-reveal-constitutive-dimerisation
#3
Ore Francis, Genevieve E Baker, Paul R Race, Josephine C Adams
The mammalian muskelin/RanBP9/CTLH complex and the S. cerevisiae GID complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), C-terminal to LisH (CTLH) and C-terminal CT11-RanBP9 (CRA) domains...
December 5, 2016: Bioscience Reports
https://www.readbyqxmd.com/read/27917836/the-crystal-structure-of-dihydrodipicolinate-reductase-from-the-human-pathogenic-bacterium-bartonella-henselae-strain-houston-1-at-2-3%C3%A2-%C3%A3-resolution
#4
Ali R Cala, Maria T Nadeau, Jan Abendroth, Bart L Staker, Alexandra R Reers, Anthony W Weatherhead, Renwick C J Dobson, Peter J Myler, André O Hudson
In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%(w/v) PEG 4000, 200 mM sodium acetate, 100 mM sodium citrate tribasic pH 5...
December 1, 2016: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/27917835/coxsackievirus-b3-protease-3c-expression-purification-crystallization-and-preliminary-structural-insights
#5
Stavroula Fili, Alexandros Valmas, Magdalini Christopoulou, Maria Spiliopoulou, Nikos Nikolopoulos, Julie Lichière, Souzana Logotheti, Fotini Karavassili, Eleftheria Rosmaraki, Andrew Fitch, Jonathan Wright, Detlef Beckers, Thomas Degen, Gwilherm Nénert, Rolf Hilgenfeld, Nicolas Papageorgiou, Bruno Canard, Bruno Coutard, Irene Margiolaki
Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0...
December 1, 2016: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/27914074/common-challenges-in-studying-the-structure-and-function-of-bacterial-proteins-case-studies-from-helicobacter-pylori
#6
Daniel A Bonsor, Eric J Sundberg
Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914071/differential-radial-capillary-action-of-ligand-assay-dracala-for-high-throughput-detection-of-protein-metabolite-interactions-in-bacteria
#7
Mona W Orr, Vincent T Lee
Bacteria rely on numerous nucleotide second messengers for signal transduction such as cyclic AMP, cyclic-di-GMP, and cyclic-di-AMP. Although a number of receptors responsible for known regulated phenotypes have been established, the completeness of protein receptors in any given organism remains elusive. We have developed a method called differential radial capillary action of ligand assay (DRaCALA) that allows for an unbiased, systematic high-throughput screen for the detection of ligand binding proteins encoded by a genome...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914046/production-of-computationally-designed-small-soluble-and-membrane-proteins-cloning-expression-and-purification
#8
Barsa Tripathy, Rudresh Acharya
This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27906451/proximity-dependent-biotinylation-for-identification-of-interacting-proteins
#9
Valerie Le Sage, Alessandro Cinti, Andrew J Mouland
Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins...
December 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27899317/neurotrophic-factor-mediated-neuronal-differentiation-of-human-cord-blood-mesenchymal-stem-cells-and-their-applicability-to-assess-the-developmental-neurotoxicity
#10
Sadaf Jahan, Dipak Kumar, Ashvini Kumar, Chetan Singh Rajpurohit, Shripriya Singh, Akriti Srivastava, Ankita Pandey, A B Pant
Plasticity and developmental capacity of stem cells have now been established as a promising tool to restore the degenerative disorders. The linearity differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, chondrogenic, osteogenic and even in neuronal subtypes has been demonstrated. The number of xenobiotics such as dexamethasone, insulin, isobutyl 1-methyle xanthine and retinoic acid has been reported for the potential to differentiate hMSCs into neuronal subtypes. But, the applicability of indigenous neurotrophic factor-nerve growth factor (NGF) has not been explored for the purpose...
November 27, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27895115/shared-subunits-of-tetrahymena-telomerase-holoenzyme-and-replication-protein-a-have-different-functions-in-different-cellular-complexes
#11
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
#12
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/27892477/nanomolar-oligomerization-and-selective-co-aggregation-of-%C3%AE-synuclein-pathogenic-mutants-revealed-by-single-molecule-fluorescence
#13
Emma Sierecki, Nichole Giles, Quill Bowden, Mark E Polinkovsky, Janina Steinbeck, Nicholas Arrioti, Diya Rahman, Akshay Bhumkar, Philip R Nicovich, Ian Ross, Robert G Parton, Till Böcking, Yann Gambin
Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps...
November 28, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27890709/role-of-major-and-brain-specific-sgce-isoforms-in-the-pathogenesis-of-myoclonus-dystonia-syndrome
#14
Jianfeng Xiao, Satya R Vemula, Yi Xue, Mohammad M Khan, Francesca A Carlisle, Adrian J Waite, Derek J Blake, Ioannis Dragatsis, Yu Zhao, Mark S LeDoux
Loss-of-function mutations in SGCE, which encodes ε-sarcoglycan (ε-SG), cause myoclonus-dystonia syndrome (OMIM159900, DYT11). A "major" ε-SG protein derived from CCDS5637.1 (NM_003919.2) and a "brain-specific" protein, that includes sequence derived from alternative exon 11b (CCDS47642.1, NM_001099400.1), are reportedly localized in post- and pre-synaptic membrane fractions, respectively. Moreover, deficiency of the "brain-specific" isoform and other isoforms derived from exon 11b may be central to the pathogenesis of DYT11...
November 24, 2016: Neurobiology of Disease
https://www.readbyqxmd.com/read/27890399/a-partially-purified-outer-membrane-protein-virb9-1-for-low-cost-nanovaccines-against-anaplasma-marginale
#15
Liang Zhao, Antonino S Cavallaro, David Wibowo, Bing Zhang, Jun Zhang, Neena Mitter, Chengzhong Yu, Chun-Xia Zhao, Anton P J Middelberg
Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice...
November 24, 2016: Vaccine
https://www.readbyqxmd.com/read/27888023/integrated-process-for-the-purification-and-immobilization-of-the-envelope-protein-domain-iii-of-dengue-virus-type-2-expressed-in-rachiplusia-nu-larvae-and-its-potential-application-in-a-diagnostic-assay
#16
María Emilia Smith, Alexandra Marisa Targovnik, Julieta Cerezo, María Alejandra Morales, María Victoria Miranda, Julián Rodríguez Talou
Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus...
November 22, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27887582/yptb3816-of-yersinia-pseudotuberculosis-strain-ip32953-is-a-virulence-related-metallo-oligopeptidase
#17
Ali Atas, Alan M Seddon, Donna C Ford, Ian A Cooper, Brendan W Wren, Petra C F Oyston, Andrey V Karlyshev
BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations...
November 25, 2016: BMC Microbiology
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
#18
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/27878624/the-creb-deubiquitinating-enzyme-does-not-directly-target-the-crea-repressor-protein-in-aspergillus-nidulans
#19
Md Ashiqul Alam, Niyom Kamlangdee, Joan M Kelly
Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism...
November 23, 2016: Current Genetics
https://www.readbyqxmd.com/read/27873257/bacterial-histidine-kinases-overexpression-purification-and-inhibitor-screen
#20
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
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