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Purification his tagged protein

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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
#1
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...
September 18, 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
#2
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...
September 6, 2017: 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
#3
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
#4
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
#5
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 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 E. 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
#6
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
#7
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
#8
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
https://www.readbyqxmd.com/read/28730304/novel-ribonuclease-activity-of-cusativin-from-cucumis-sativus-for-mapping-nucleoside-modifications-in-rna
#9
Balasubrahmanyam Addepalli, Sarah Venus, Priti Thakur, Patrick A Limbach
A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein...
July 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28706572/a-microwell-printing-fabrication-strategy-for-the-on-chip-templated-biosynthesis-of-protein-microarrays-for-surface-plasmon-resonance-imaging
#10
Gerald Manuel, Andrej Lupták, Robert M Corn
A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein...
September 22, 2016: Journal of Physical Chemistry. C, Nanomaterials and Interfaces
https://www.readbyqxmd.com/read/28698826/expression-refolding-and-spectroscopic-characterization-of-fibronectin-type-iii-fniii-homology-domains-derived-from-human-fibronectin-leucine-rich-transmembrane-protein-flrt-1-2-and-3
#11
Lila Yang, Maria Hansen Falkesgaard, Peter Waaben Thulstrup, Peter Schledermann Walmod, Leila Lo Leggio, Kim Krighaar Rasmussen
The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans-interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis-interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation...
2017: PeerJ
https://www.readbyqxmd.com/read/28685146/his-flag-tag-as-a-fusion-partner-of-glycosylated-human-interferon-gamma-and-its-mutant-gain-or-loss
#12
Elena Krachmarova, Milena Tileva, Elena Lilkova, Peicho Petkov, Klaus Maskos, Nevena Ilieva, Ivan Ivanov, Leandar Litov, Genoveva Nacheva
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28670287/preparative-sds-page-as-an-alternative-to-his-tag-purification-of-recombinant-amelogenin
#13
Claire M Gabe, Steven J Brookes, Jennifer Kirkham
Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28668496/generation-of-nanobodies-against-slyd-and-development-of-tools-to-eliminate-this-bacterial-contaminant-from-recombinant-proteins
#14
Yaozhong Hu, Ema Romão, Didier Vertommen, Cécile Vincke, Francisco Morales-Yánez, Carlos Gutiérrez, Changxiao Liu, Serge Muyldermans
The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response...
September 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28661426/rational-design-of-recombinant-papain-like-cysteine-protease-optimal-domain-structure-and-expression-conditions-for-wheat-derived-enzyme-triticain-%C3%AE
#15
Neonila V Gorokhovets, Vladimir A Makarov, Anastasiia I Petushkova, Olga S Prokopets, Mikhail A Rubtsov, Lyudmila V Savvateeva, Evgeni Yu Zernii, Andrey A Zamyatnin
Triticain-α is a papain-like cysteine protease from wheat (Triticumaestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones...
June 29, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28628836/an-affinity-based-aqueous-two-phase-mixed-micellar-system-and-its-purification-of-yeast-3-5-bisphosphate-nucleotidase
#16
Yao Liu, Mei-Hao Sun, Shi-Kuan Shao, Gang Deng
Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system...
August 15, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28625912/isolation-of-recombinant-human-untagged-glyceraldehyde-3-phosphate-dehydrogenase-from-e-%C3%A2-coli-producer-strain
#17
K V Barinova, M A Eldarov, E V Khomyakova, V I Muronetz, E V Schmalhausen
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass)...
September 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624997/expression-and-purification-of-cytochrome-p450-55b1-from-chlamydomonas-reinhardtii-and-its-application-in-nitric-oxide-biosensing
#18
Bin Gong, Xiaosheng Liang, Yong Li, Qian Xiao, Panchun Yang, Yunhua Wu
Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0...
June 17, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28624493/expression-of-the-c-terminal-domain-of-human-apolipoprotein-a-i-using-a-chimeric-apolipoprotein
#19
Daniel E Sallee, James V C Horn, Lukas A Fuentes, Paul M M Weers
Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification...
September 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28575696/regiospecific-radiolabelling-of-nanofitin-on-ni-magnetic-beads-with-18-f-fbem-and-in-vivo-pet-studies
#20
Sylvestre Dammicco, Marine Goux, Christian Lemaire, Guillaume Becker, Mohamed Ali Bahri, Alain Plenevaux, Mathieu Cinier, André Luxen
INTRODUCTION: Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs...
April 27, 2017: Nuclear Medicine and Biology
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