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Purification his tagged protein

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https://www.readbyqxmd.com/read/29345069/automation-aided-optimization-of-cloning-expression-and-purification-of-enzymes-of-the-bacterial-sialic-acid-catabolic-and-sialylation-pathways-enzymes-for-structural-studies
#1
Sneha Bairy, Lakshmi Narayanan Gopalan, Thanuja Gangi Setty, Sathya Srinivasachari, Lavanyaa Manjunath, Jay Prakash Kumar, Sai R Guntupalli, Sucharita Bose, Vinod Nayak, Swagatha Ghosh, Nitish Sathyanarayanan, Rhawnie Caing-Carlsson, Weixiao Yuan Wahlgren, Rosmarie Friemann, S Ramaswamy, Muniasamy Neerathilingam
The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway...
January 17, 2018: Microbial Biotechnology
https://www.readbyqxmd.com/read/29340583/functional-analysis-of-the-helicobacter-pullorum-n-linked-protein-glycosylation-system
#2
Adrian J Jervis, Alison G Wood, Joel A Cain, Jonathan A Butler, Helen Frost, Elizabeth Lord, Rebecca Langdon, Stuart J Cordwell, Brendan W Wren, Dennis Linton
N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H...
January 11, 2018: Glycobiology
https://www.readbyqxmd.com/read/29318540/identification-of-proteolytic-cleavage-sites-of-epha2-by%C3%A2-membrane-type-1-matrix-metalloproteinase-on-the%C3%A2-surface-of-cancer-cells
#3
Keiji Kikuchi, Hiroko Kozuka-Hata, Masaaki Oyama, Motoharu Seiki, Naohiko Koshikawa
Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification ...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29287247/aptamer-facilitated-purification-of-functional-proteins
#4
Stanislav S Beloborodov, Jiayin Bao, Svetlana M Krylova, Agnesa Shala-Lawrence, Philip E Johnson, Sergey N Krylov
DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state...
December 16, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29242048/simplified-immobilisation-method-for-histidine-tagged-enzymes-in-poly-methyl-methacrylate-microfluidic-devices
#5
Gulsim Kulsharova, Nikolay Dimov, Marco P C Marques, Nicolas Szita, Frank Baganz
Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker...
December 11, 2017: New Biotechnology
https://www.readbyqxmd.com/read/29229289/expression-of-fibronectin-binding-protein-of-l-acidophilus-ncfm-and-in-vitro-refolding-to-adhesion-capable-native-like-protein-from-inclusion-bodies
#6
Sonu Bisht, Kumar Siddharth Singh, Ritu Choudhary, Sudarshan Kumar, Sunita Grover, Ashok Kumar Mohanty, Veena Pande, Jai Kumar Kaushik
The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form...
December 8, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29223155/recombinant-human-erythropoietin-with-additional-processable-protein-domains-purification-of-protein-synthesized-in-escherichia-coli-heterologous-expression-system
#7
T M Grunina, A V Demidenko, A M Lyaschuk, M S Poponova, Z M Galushkina, L A Soboleva, S A Cherepushkin, N B Polyakov, D A Grumov, A I Solovyev, V G Zhukhovitsky, I S Boksha, M E Subbotina, A V Gromov, V G Lunin, A S Karyagina
Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible...
November 2017: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29213997/cloning-optimization-of-induction-conditions-and-purification-of-mycobacterium-tuberculosis-rv1733c-protein-expressed-in-escherichia-coli
#8
Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph
Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E...
April 2017: Iranian Journal of Microbiology
https://www.readbyqxmd.com/read/29203835/purification-and-functional-comparison-of-nine-human-aquaporins-produced-in-saccharomyces-cerevisiae-for-the-purpose-of-biophysical-characterization
#9
Frederik Bühring Bjørkskov, Simon Lyngaa Krabbe, Casper Normann Nurup, Julie Winkel Missel, Mariana Spulber, Julie Bomholt, Karen Molbaek, Claus Helix-Nielsen, Kamil Gotfryd, Pontus Gourdon, Per Amstrup Pedersen
The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets...
December 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29202520/-expression-and-purification-of-rabies-virus-glycoprotein-and-analysis-of-its-specific-binding-capacity-to-memory-b-cells
#10
Liwei Yan, Wei Gong, Wenbing Zhu, Xuemei Zhang, Jingwen Xu, Zhongxiang Wu, Kongjie Lu, Ming Sun, Shaozhong Dong
We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/29193968/structural-insight-into-the-photochemistry-of-split-green-fluorescent-proteins-a-unique-role-for-a-his-tag
#11
Alan Deng, Steven G Boxer
Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e. split GFP with a β-strand removed, that were found to behave differently in the presence of light...
December 1, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29161226/development-of-competitive-enzyme-linked-immunosorbent-assays-for-antibody-detection-based-on-bluetongue-virus-monoclonal-antibodies
#12
Zhou Han, Zang Mingxin, Li Xuechun, Xu Yigang, Qiao Xinyuan, Wang Li, Cui Wen, Jiang Yanping, Li Yijing, Tang Lijie
Bluetongue is a ruminant infectious disease that is characterized by hyperpyrexia, leukocyte decrease and mucosal ulcerative inflammation changes. In this study, three segments of Bluetongue virus (BTV)-8 VP2 protein (BTV-8A, 8B, and 8C) were cloned into pET-28a (+) and pMAl-c5X vectors and expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-8A/8B/8C) and maltose-binding protein (MBP)-tagged (MBP-8A/8B/8C) fusion proteins. After purification, His-8A/8B/8C were used to immunize BALB/mice and MBP-8A/8B/8C were used to screen for monoclonal antibody (mAb)-secreting hybridomas...
November 21, 2017: Viral Immunology
https://www.readbyqxmd.com/read/29154996/selective-binding-magnetic-separation-and-purification-of-histidine-tagged-protein-using-biopolymer-magnetic-core-shell-nanoparticles
#13
Yang Zhou, Dandan Yan, Shaofei Yuan, Yawen Chen, Emmanuella E Fletcher, Haifeng Shi, Bangxing Han
In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni(2+) nanoparticles. The Ni(2+) on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nanoparticles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified...
November 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29149997/surface-sieving-coordinated-imac-material-for-purification-of-his-tagged-proteins
#14
Senwu Li, Kaiguang Yang, Lukuan Liu, Baofeng Zhao, Yuanbo Chen, Xiao Li, Lihua Zhang, Yukui Zhang
Tailor-made materials for the purification of proteins with His-tag was designed through synergizing the selectivity of surface sieving and metal ion affinity. By excluding impurity proteins out of the surface polymer network, such materials could purify His-tagged proteins from the crude cell lysis with purity up to 90%, improved by 14% compared to that obtained by the commercial metal chelating affinity materials. This study might promote the His-tagged protein purification to a new level.
January 2, 2018: Analytica Chimica Acta
https://www.readbyqxmd.com/read/29146152/improved-antifungal-activity-of-barley-derived-chitinase-i-gene-that-overexpress-a-32kda-recombinant-chitinase-in-escherichia-coli-host
#15
Nida Toufiq, Bushra Tabassum, Muhammad Umar Bhatti, Anwar Khan, Muhammad Tariq, Naila Shahid, Idrees Ahmad Nasir, Tayyab Husnain
Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene...
October 31, 2017: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
https://www.readbyqxmd.com/read/29116178/alternative-application-of-an-affinity-purification-tag-hexahistidines-in-ester-hydrolysis
#16
Lise Schoonen, Kayleigh S van Esterik, Chunqiu Zhang, Rein V Ulijn, Roeland J M Nolte, Jan C M van Hest
Hexahistidines are very common tags used in the affinity chromatography purification of recombinant proteins. Although these tags are solely applied for their metal-binding properties, we found that they are also able to perform ester hydrolysis when attached to a protein. For instance, green fluorescent protein (GFP) and the cowpea chlorotic mottle virus (CCMV) are able to perform catalysis after introduction of the His-tag. By attaching a His-tag to an enzyme, a dual-functional catalyst was created, that can perform a two-step cascade reaction...
November 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29111374/layer-by-layer-coating-of-nh2-silicate-polycarboxylic-acid-polymer-saturated-by-ni-2-onto-the-super-magnetic-nife2o4-nanoparticles-for-sensitive-and-bio-valuable-separation-of-his-tagged-proteins
#17
Seyede Zohreh Mirahmadi-Zare, Fatemeh Aboutalebi, Maryam Allafchian, Leila Pirjamali, Mohammad-Hossein Nasr-Esfahani
Magnetic nanoparticles NiFe2O4 was synthesized and covered in the silicate lattice of (3-Aminopropyl) triethoxysilane (APS) by the sol-gel process. Subsequently, the EDTA-dianhydride was attached to the amino surface of magnetic nanoparticles (MNPs) during the nucleophilic attack. This polycarboxylic layer trapped the high level of nickel ions for selective bonding to the His-tagged recombinant protein. The surface of MNPs was investigated by TEM, XRD, SEM (EDSA), VSM, BET, FT-IR and zeta potential analysis which characterized the size, chemical lattice, morphology, magnetic strength, specific surface area, functional groups and charge of the surface of nanoparticles...
October 28, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29063721/high-yield-purification-and-first-structural-characterization-of-the-full-length-bacterial-toxin-cnf1
#18
Andrea Colarusso, Marco Caterino, Alessia Fabbri, Carla Fiorentini, Alessandro Vergara, Filomena Sica, Ermenegilda Parrilli, Maria Luisa Tutino
The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborisation, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies...
October 24, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/29062201/cloning-and-expression-of-p67-protein-of-mycoplasma-leachii
#19
Sabarinath Thankappan, Rajneesh Rana, Arun Thachappully Remesh, Valsala Rekha, Viswas Konasagara Nagaleekar, Bhavani Puvvala
AIM: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii. MATERIALS AND METHODS: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography...
September 2017: Veterinary World
https://www.readbyqxmd.com/read/29061519/a-novel-reverse-micellar-purification-strategy-for-histidine-tagged-human-interferon-gamma-hifn-%C3%AE-protein-from-pichia-pastoris
#20
Ashish A Prabhu, Anwesha Purkayastha, Bapi Mandal, Jadi Praveen Kumar, Biman B Mandal, V Venkata Dasu
In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ was cloned in Pichia pastoris and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE)...
October 20, 2017: International Journal of Biological Macromolecules
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