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Protein expression and purification

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https://www.readbyqxmd.com/read/28338731/purification-and-enzymatic-characterization-of-gallus-gallus-blm-helicase
#1
Jing Shi, Na-Nv Liu, Yan-Tao Yang, Xu-Guang Xi
Mutations in human BLM helicase give rise to the autosomal recessive Bloom syndrome, which shows high predisposition to types of malignant tumours. Though lots of biochemical and structural investigations have shed lights on the helicase core, structural investigations of the whole BLM protein are still limited due to its low stability and production. Here by comparing with the expression systems and functions of other BLM homologues, we developed the heterologous high-level expression and high-yield purification systems for Gallus gallus BLM (gBLM) in Escherichia coli...
February 21, 2017: Journal of Biochemistry
https://www.readbyqxmd.com/read/28338200/glioma-cells-promote-angiogenesis-through-the-release-of-exosomes-containing-long-non-coding-rna-pou3f3
#2
H-L Lang, G-W Hu, Y Chen, Y Liu, W Tu, Y-M Lu, L Wu, G-H Xu
OBJECTIVE: Angiogenesis is a key event in the progression of gliomas, and emerging evidence suggests that exosomes are signaling extracellular organelles that modulate the tumor microenvironment and promote angiogenesis and tumor progression. This study aimed to explore the mechanism by which glioma-derived exosomes affect angiogenesis. MATERIALS AND METHODS: qRT-PCR was used to determine the expression level of linc-POU3F3 in glioma tissue as well as glioma cell lines...
March 2017: European Review for Medical and Pharmacological Sciences
https://www.readbyqxmd.com/read/28334977/sclip-an-integrated-platform-to-study-rna-protein-interactomes-in-biomedical-research-identification-of-cstf2tau-in-alternative-processing-of-small-nuclear-rnas
#3
Yulia Kargapolova, Michal Levin, Karl Lackner, Sven Danckwardt
RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here, we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing...
February 28, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28330749/identification-purification-and-expression-patterns-of-chitinase-from-psychrotolerant-pedobacter-sp-pr-m6-and-antifungal-activity-in-vitro
#4
Yong-Su Song, Dong-Jun Seo, Woo-Jin Jung
In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C...
March 19, 2017: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28330270/expression-and-purification-of-a-gene-encoding-a-9-7%C3%A2-kda-pe-protein-of-mycobacterium-avium-subsp-paratuberculosis
#5
S Chandra Sekar, P P Goswami, R Deb
Mycobacterium avium subsp. paratuberculosis (Map) contains PE family antigens which are Proline and glutamic acid rich and may play important role as T-cell antigens. In the present study, the Map 1507 ORF encoding 9.7 kDa PE protein was amplified by polymerase chain reaction and cloned into E. coli vector pQE30 UA. The recombinant plasmid designated as pQ PE was transformed into E. coli M15 cells and induced with IPTG revealed the high level expression of 11.9 kDa His-fusion protein as estimated by migration in 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330257/generation-of-an-inducible-system-to-express-polo-like-kinase-cdc5-as-tap-fusion-protein-during-meiosis-in-saccharomyces-cerevisiae
#6
Rajni Vaid, Kamal Dev, Michael Lichten, Anuradha Sourirajan
Tandem affinity purification (TAP) is a highly efficient method for isolation of protein complexes from endogenous biological macromolecules. TAP system consists of dual affinity tags that facilitates the sequential purification of the desired proteins expressed at their low levels in vivo. Polo-like kinases (PLK) are serine/threonine protein kinases that are the crucial regulators of cell cycle. Cdc5, the solitary PLK in budding yeast Saccharomyces cerevisiae, has diverse array of targets in cell cycle. The present study was undertaken to construct an estrogen-inducible system for expression of Cdc5-TAP to isolate the substrates of Cdc5 during meiosis, particularly, pachytene stage of meiosis I...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330241/purification-characterization-gene-cloning-and-expression-of-gh-10-xylanase-penicillium-citrinum-isolate-hzn13
#7
Zabin K Bagewadi, Sikandar I Mulla, Harichandra Z Ninnekar
An extracellular thermostable xylanase (Xyl-IIb) produced by Penicillium citrinum isolate HZN13 was purified to homogeneity using DEAE-Sepharose, Sephadex G-100 and Bio-Gel P-60 chromatography with specific activity of 6272.7 U/mg and 19.6-fold purification. The purification revealed the occurrence of multiple forms of xylanases (Xyl-I, Xyl-IIa, Xyl-IIb and Xyl-III). The molecular mass of highly purified Xyl-IIb was ~31 kDa with SDS-PAGE. The enzyme was cellulase-free, thermostable (55-75 °C) and acidophilic (3...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330196/cloning-and-expression-of-saccharomyces-cerevisiae-suc2-gene-in-yeast-platform-and-characterization-of-recombinant-enzyme-biochemical-properties
#8
Nooshin Mohandesi, Seyed Omid Ranaei Siadat, Kamahldin Haghbeen, Ardeshir Hesampour
Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330118/cloning-expression-purification-and-characterization-of-lipase-from-bacillus-licheniformis-isolated-from-hot-spring-of-himachal-pradesh-india
#9
Gagandeep Kaur, Amninder Singh, Rohit Sharma, Vinay Sharma, Swati Verma, Pushpender K Sharma
In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg(-1) and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i...
June 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330113/protein-fusion-tags-for-efficient-expression-and-purification-of-recombinant-proteins-in-the-periplasmic-space-of-e-coli
#10
REVIEW
Ajamaluddin Malik
Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical...
June 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330086/cloning-expression-purification-and-characterization-of-human-mitochondrial-carbonic-anhydrase-va
#11
Danish Idrees, Sudhir Kumar, Syed Abdul Arif Rehman, Samudrala Gourinath, Asimul Islam, Faizan Ahmad, Md Imtaiyaz Hassan
Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO2 to produce HCO3(-) and proton. CAV is primarily involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis by providing bicarbonate ion. Here, we report a new strategy for cloning, expression and purification for CAVA in the bacterial system followed by its biophysical characterization. The cDNA of CAVA, a 801 nucleotide long that encodes a 267-amino acid polypeptide of molecular mass of 30-kDa (excluding signal peptide), was sub-cloned in the expression vector pET21c and transformed into Escherichia coli strain BL21 (DE3) for expression...
June 2016: 3 Biotech
https://www.readbyqxmd.com/read/28328957/differential-effects-of-deae-negative-mode-chromatography-and-gel-filtration-chromatography-on-the-charge-status-of-helicobacter-pylori-neutrophil-activating-protein
#12
Zhi-Wei Hong, Yu-Chi Yang, Timothy Pan, Huey-Fen Tzeng, Hua-Wen Fu
Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification...
2017: PloS One
https://www.readbyqxmd.com/read/28326614/recombinant-expression-of-porcine-spermadhesin-awn-and-its-phospholipid-interaction-indication-for-a-novel-lipid-binding-property
#13
F Schröter, K Müller, P Müller, E Krause, B C Braun
AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity...
March 21, 2017: Reproduction in Domestic Animals, Zuchthygiene
https://www.readbyqxmd.com/read/28323416/one-pot-two-nanoprobe-assay-uncovers-targeted-glycoprotein-biosignature
#14
Mira Anne C Dela Rosa, Wei-Chun Chen, Yi-Ju Chen, Rofeamor P Obena, Chih-Hsiang Chang, Rey Y Capangpangan, Tung-Hung Su, Chi-Ling Chen, Pei-Jer Chen, Yu-Ju Chen
We report a one-pot two-nanoprobe approach coupled to mass spectrometry for simultaneous quantification and post-translational modification (PTM) profiling of targeted protein in biofluid. Using N-glycoprotein as model, the assay employs two nanoprobes, antibody-conjugated SiO2 nanoparticles and lectin-conjugated magnetic Fe3O4 nanoparticles, to achieve target glycoprotein isolation from biofluid and subsequent glycopeptide enrichment in a single tube. As demonstrated on α-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma (HCC), the assay has high purification specificity (20 glycopeptides) with 2-fold and 10-fold superior total glycopeptide intensity compared to non-one-pot method (9 glycopeptides) or without enrichment (6 glycopeptides), respectively...
March 21, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28323169/isolation-and-characterization-of-human-capg-expressed-and-post-translationally-modified-in-pichia-pastoris
#15
Agnes Papala, Marc Sylvester, Nadine Dyballa-Rukes, Sabine Metzger, Jochen D'Haese
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification...
March 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28323168/production-and-characterization-of-a-highly-pure-rna-polymerase-holoenzyme-from-mycobacterium-tuberculosis
#16
Omar Herrera-Asmat, Lucyna Lubkowska, Mikhail Kashlev, Carlos J Bustamante, Daniel G Guerra, Maria L Kireeva
Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies...
March 17, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28322781/characterization-of-akr1b16-a-novel-mouse-aldo-keto-reductase
#17
Joan Giménez-Dejoz, Susanne Weber, Oleg A Barski, Gabriele Möller, Jerzy Adamski, Xavier Parés, Sergio Porté, Jaume Farrés
Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs...
March 17, 2017: Chemico-biological Interactions
https://www.readbyqxmd.com/read/28320377/application-of-an-e-coli-signal-sequence-as-a-versatile-inclusion-body-tag
#18
Wouter S P Jong, David Vikström, Diane Houben, H Bart van den Berg van Saparoea, Jan-Willem de Gier, Joen Luirink
BACKGROUND: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form...
March 21, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28319379/immobilization-of-%C3%AE-galactosidases-from-lactobacillus-on-chitin-using-chitin-binding-domain
#19
Mai Lan Mai Pham, Tatjana Leister, Hoang Anh Nguyen, Bien-Cuong Do, Tuan-Anh Pham, Dietmar Haltrich, Montarop Yamabhai, Thu-Ha Nguyen, Tien-Thanh Nguyen
Two β-galactosidases from Lactobacillus including a heterodimeric, LacLM type enzyme from L. reuteri L103 and a homodimeric LacZ type β-galactosidase from L. bulgaricus DSM 20081 were studied for immobilization on chitin using a carbohydrate-binding domain (chitin-binding domain, ChBD) from a chitinolytic enzyme. Three recombinant enzymes, namely LacLM-ChBD, ChBD-LacLM and LacZ-ChBD, were constructed and successfully expressed in L. plantarum WCFS1. Depending on the structure of the enzymes, either homodimeric or heterodimeric, as well as the positioning of the chitin-binding domain in relation to the catalytic domains, i...
March 20, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28317180/anti-acsa-2-defines-a-novel-monoclonal-antibody-for-prospective-isolation-of-living-neonatal-and-adult-astrocytes
#20
Christina G Kantzer, Camille Boutin, Ina D Herzig, Carolina Wittwer, Sandy Reiß, Marie Catherine Tiveron, Jan Drewes, Thomas D Rockel, Stefanie Ohlig, Jovica Ninkovic, Harold Cremer, Sandra Pennartz, Melanie Jungblut, Andreas Bosio
Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2(+) cells, microglia, endothelial cells, leukocytes, or erythrocytes...
March 20, 2017: Glia
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