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Purification his tagged protein

Mingcong Shao, Lili Xiu, Haijiang Zhang, Jianying Huang, Xingwen Gong
Chitosan/cellulose-based beads (CCB) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as solvent, and their structures were characterized by fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and thermo gravimetric analysis (TGA). The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli (E. coli). The results showed that Zn2 + -CCB exhibited more specific adsorption capacity towards the target protein compared with Ni2 + -CCB and Cu2 + -CCB...
March 6, 2018: Preparative Biochemistry & Biotechnology
Prasannan V Anu, Madathiparambil G Madanan, Ananthakrishnan J Nair, Gangaprasad A Nair, Govinda Pillai M Nair, Perumana R Sudhakaran, Padikara K Satheeshkumar
Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag)...
March 3, 2018: Molecular Biotechnology
James R O Eaton, Yara Alenazi, Kamayani Singh, Graham Davies, Lucia Geis-Asteggiante, Benedikt Kessler, Carol V Robinson, Akane Kawamura, Shoumo Bhattacharya
Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and have shown anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus. We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nM and four other CC chemokines with a Kd between 10 and 100 nM, and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nM. The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8...
February 27, 2018: Journal of Biological Chemistry
Aiganym T Zhumabek, Laura S Abeuova, Nurzhan S Mukhametzhanov, Herman B Scholthof, Yerlan M Ramankulov, Shuga A Manabayeva
Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle...
February 1, 2018: Journal of Virological Methods
Vishwa D Trivedi, Tashmay S Jones, Renee P Walker
Advances in biotechnology generated wide range of microbial genome and their related protein database. Freshwater cyanobacterium Anabaena PCC7120 sensory rhodopsin, ASR in contrast to classical haloarchaeal sensory rhodopsins interacts with putative soluble transducer, ASRT. The 125 amino acid transducer exists as a soluble protein and is involved in photoreceptor binding. Recombinant DNA tools in biotechnology conventionally support the use of affinity tags for ease of protein purification and subsequent studies...
August 2017: Int J Eng Sci (Ghaziabad)
Vaishali Verma, Charanpreet Kaur, Payal Grover, Amita Gupta, Vijay K Chaudhary
The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro...
2018: PloS One
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
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
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
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
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
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...
May 2018: Protein Expression and Purification
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
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
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
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
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...
January 10, 2018: Journal of the American Chemical Society
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
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 Fe3 O4 /PMG/IDA-Ni2+ nanoparticles. The Ni2+ 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 (Fe3 O4 ) 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...
April 2018: Protein Expression and Purification
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
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