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Purification his tagged protein

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https://www.readbyqxmd.com/read/29116178/alternative-application-of-an-affinity-purification-tag-hexahistidines-in-ester-hydrolysis
#1
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
#2
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
#3
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
#4
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
#5
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
https://www.readbyqxmd.com/read/29032467/a-helicobacter-pylori-vacuolating-cytotoxin-a-mouse-dhfr-fusion-protein-triggers-dye-release-from-liposomes
#6
Aung Khine Linn, Nitchakan Samainukul, Somsri Sakdee, Chanan Angsuthanasombat, Gerd Katzenmeier
The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E...
October 14, 2017: Current Microbiology
https://www.readbyqxmd.com/read/29027449/-soluble-expression-purification-and-structural-analysis-of-the-bhlh-transcription-factor-bmsage-of-bombyx-mori
#7
Huawei He, Shuguang Wei, Yejing Wang, Lina Liu, Zhenzhen Li, Peng Zhao, Huaipu Chang, Ping Zhao
Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/29026495/recombinant-production-and-affinity-purification-of-the-frac-pore-forming-toxin-using-hexa-his-tag-and-pet-expression-cassette
#8
Mehdi Imani, Hossein Zarei Jaliani, Mohammad Hassan Kheirandish, Mahnaz Azadpour
OBJECTIVES: A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification. MATERIALS AND METHODS: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+)...
April 2017: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/28971274/a-lactococcus-lactis-expression-vector-set-with-multiple-affinity-tags-to-facilitate-isolation-and-direct-labeling-of-heterologous-secreted-proteins
#9
Francisco Romero Pastrana, Jolanda Neef, Jan Maarten van Dijl, Girbe Buist
The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His6)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling...
November 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28963004/high-level-expression-of-soluble-recombinant-proteins-in-escherichia-coli-using-an-he-maltotriose-binding-protein-fusion-tag
#10
Yingqian Han, Wanying Guo, Bingqian Su, Yujie Guo, Jiang Wang, Beibei Chu, Guoyu Yang
Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli...
September 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28950808/engineering-of-an-episomal-plasmid-suitable-for-high-throughput-expression-in-pichia-pastoris
#11
Huan Chen, Cui Cao, Anna Kulinich, Li Liu, Yong-Sam Jung, Josef Voglmeir
AIM AND OBJECTIVE: This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB. MATERIAL AND METHOD: The performance of pPICZαBHF was evaluated with red fluorescence protein as a reporter. Additional His- and Flag-tags on the N-terminal ensure a simplified protein purification procedure. Transformation efficiency, expression level and plasmid maintenance were studied in order to test the functionality and usefulness of the constructed vector...
September 25, 2017: Combinatorial Chemistry & High Throughput Screening
https://www.readbyqxmd.com/read/28941789/streamlined-purification-of-fluorescently-labeled-escherichia-coli-phosphate-binding-protein-phos-suitable-for-rapid-kinetics-applications
#12
Dustin D Smith, Dylan Girodat, Hans-Joachim Wieden, L Brent Selinger
Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (Pi) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye...
November 15, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28921304/expression-and-purification-of-an-fgf9-fusion-protein-in-e-coli-and-the-effects-of-the-fgf9-subfamily-on-human-hepatocellular-carcinoma-cell-proliferation-and-migration
#13
Shen Wang, Haipeng Lin, Tiantian Zhao, Sisi Huang, David G Fernig, Nuo Xu, Fenfang Wu, Mi Zhou, Chao Jiang, Haishan Tian
Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity...
November 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28888757/single-step-purification-of-recombinant-gaussia-luciferase-from-serum-containing-culture-medium-of-mammalian-cells
#14
Satoshi Inouye
A dihydrofolate reductase-deficient Chinese hamster ovary (CHO-K1/dhfr(-)) cell line stably expressing Gaussia luciferase with a histidine-tag sequence at the carboxyl terminus (GLase-His) was established. Recombinant GLase-His was purified from serum-containing culture medium by single-step Ni-chelate column chromatography in the presence of 2 M NaCl and 0.01% Tween 20. The protein yield of GLase-His with over 95% purity was 0.5 mg from 0.9 L of the cultured medium. The enzymatic properties of purified GLase-His were characterized...
January 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/28866214/facile-fabrication-of-nickel-immobilized-on-magnetic-nanoparticles-as-an-efficient-affinity-adsorbent-for-purification-of-his-tagged-protein
#15
Zahra Rashid, Hossein Naeimi, Amir-Hassan Zarnani, Fereshteh Mohammadi, Ramin Ghahremanzadeh
In the present research, an efficient, convenient, and inexpensive method for the one-pot synthesis of Fe3O4@Histidine is developed. Histidine is readily loaded on magnetic nanoparticles by one step and simple method without any supplemental linkers. In the structure of Fe3O4@Histidine, histidine covalently immobilized on the surface of Fe3O4, magnetic nanoparticles are able to trap Ni(2+) ions through a strong interaction between nickel and histidines in protein tag. Two coordination sites of nickel are occupied with ligand on the surface of magnetic nanoparticles and four coordination sites have been remained that these sites will be occupied with histidine tag of recombinant protein A...
November 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/28832667/secretory-expression-and-purification-of-bacillus-licheniformis-keratinase-in-insect-cells
#16
Miaorong Huang, Ruiai Chen, Guangcai Ren
The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ cell to generate recombinant bacmid carrying Ker-His-Flag...
2017: PloS One
https://www.readbyqxmd.com/read/28816641/preparation-of-recombinant-human-thymic-stromal-lymphopoietin-protein
#17
Yanlu Zhang, Liehua Wu, Jingai Yu, JianFeng Mei, Yu Yi, JianShu Chen, Guoqing Ying
Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification...
August 17, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28768815/synthesis-and-self-assembly-of-cellulose-microfibrils-from-reconstituted-cellulose-synthase
#18
Sung Hyun Cho, Pallinti Purushotham, Chao Fang, Cassandra Maranas, Sara M Díaz-Moreno, Vincent Bulone, Jochen Zimmer, Manish Kumar, B Tracy Nixon
Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5...
September 2017: Plant Physiology
https://www.readbyqxmd.com/read/28761057/development-of-series-of-affinity-tags-in-streptomyces
#19
Xu-Ming Mao, Ning Sun, Yang Zheng, Yong-Quan Li
Streptomyces are of great biological and industrial significance due to their complex morphological development and ability to produce numerous secondary metabolites. However, the intrinsic biochemical mechanisms underlying morphogenesis and secondary metabolism are rarely revealed, partially because of the limited availability of the biochemical tools in Streptomyces. Here we provided series of integrative vectors with various affinity tags, including single tags 3×FLAG, 3×HA, 3×Strep-tag II, 18×His, 13×Myc, and dual tags, all of which were driven from a strong constitutive promoter ermEp*...
July 31, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28747746/one-step-purification-and-immobilization-of-extracellularly-expressed-sortase-a-by-magnetic-particles-to-develop-a-robust-and-recyclable-biocatalyst
#20
Xinrui Zhao, Haofei Hong, Xiaozhong Cheng, Shaozhong Liu, Tao Deng, Zhongwu Guo, Zhimeng Wu
Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels...
July 26, 2017: Scientific Reports
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