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Affinity chromatography

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https://www.readbyqxmd.com/read/28342583/affinity-chromatographic-methodologies-based-on-immobilized-voltage-dependent-anion-channel-isoform-1-and-application-in-protein-ligand-interaction-analysis-and-bioactive-compounds-screening-from-traditional-medicine
#1
Qian Li, Pan Qiao, Xiu Chen, Jing Wang, Liujiao Bian, Xiaohui Zheng
Voltage dependent anion channel isoform 1 (VDAC-1) serves as an attractive target of anti-cancer drugs by mediating the entry and exit of metabolites between cytoplasm and mitochondria. This work reports on the preparation of a VDAC-1-based bioaffinity chromatographic stationary phase by linking the protein on lecithin modified microspheres. An assay of chromatographic methods including frontal analysis, zonal elution, injection dependent analysis and nonlinear chromatography were utilized to investigate the bindings of ATP, NADH and NADPH to VDAC-1...
March 18, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28342173/metal-ions-binding-t4-lysozyme-as-an-intramolecular-protein-purification-tag-compatible-with-x-ray-crystallography
#2
Evzen Boura, Adriana Baumlova, Dominika Chalupska, Anna Dubankova, Martin Klima
Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag...
March 25, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28342149/identification-and-preparation-of-a-novel-chemokine-receptor-binding-domain-in-the-cytoplasmic-regulator-frount
#3
Akihiro Sonoda, Sosuke Yoshinaga, Kaori Yunoki, Soichiro Ezaki, Kotaro Yano, Mitsuhiro Takeda, Etsuko Toda, Yuya Terashima, Kouji Matsushima, Hiroaki Terasawa
FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors...
March 24, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28341915/in-vivo-system-for-analyzing-the-function-of-the-psbp-protein-using-chlamydomonas-reinhardtii
#4
Taishi Nishimura, Fumihiko Sato, Kentaro Ifuku
The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP...
March 24, 2017: Photosynthesis Research
https://www.readbyqxmd.com/read/28340986/design-and-synthesis-of-affinity-chromatography-ligands-for-the-purification-of-5-hydroxyeicosanoid-dehydrogenase
#5
Chintam Nagendra Reddy, Qiuji Ye, Pranav Patel, Sashikala Sivendran, Shishir Chourey, Rui Wang, Jaganmohan R Anumolu, Gail E Grant, William S Powell, Joshua Rokach
Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles...
January 1, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28340702/selective-solid-phase-extraction-of-jwh-synthetic-cannabinoids-by-using-computationally-designed-peptides
#6
Marcello Mascini, Camilla Montesano, German Perez, Joseph Wang, Dario Compagnone, Manuel Sergi
The objective of the present work is to demonstrate a rational way to prepare selective sorbents able to extract simultaneously several structural analogs. For this purpose the binding specificity of two hexapeptides computationally designed (VYWLVW and YYIGGF) versus four synthetic cannabinoids Naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH 018), naphthalen-1-yl-(1-butylindol-3-yl)methanone (JWH 073), (R)-(1-((1-methylpiperidin-2-yl)methyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM 1220) and (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55) was computationally studied and then experimentally tested by solid-phase extraction (SPE) clean-up and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis...
May 15, 2017: Talanta
https://www.readbyqxmd.com/read/28339033/role-of-tropomyosin-in-silkworm-allergy
#7
Kyoung Yong Jeong, In-Soo Han, June Yong Lee, Kyung Hee Park, Jae-Hyun Lee, Jung-Won Park
Silkworm pupae are widely consumed in Asian countries and allergic reactions following consumption have been described. However, false‑positive responses in skin prick allergy tests or non‑specific immunoglobulin E (IgE) responses to total extract of silkworm pupa make diagnosis difficult. Although improved allergy diagnosis is required, molecular characterization of silkworm allergens has not been performed to date, except for Bomb m 1, an arginine kinase. This study aimed to evaluate the allergenicity of tropomyosin, a well‑established invertebrate pan‑allergen, from silkworm pupa...
March 24, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28338341/natural-product-inhibitors-of-carbonic-anhydrase-i-and-ii-isoenzymes-osajin-and-pomiferin
#8
Esra Dilek, Hüseyin Serkan Erol, Ahmet Cakir, Murat Koc, Mesut Bünyami Halici
The aim of this study is to purify carbonic anhydrase I and II isoenzymes from human erythrocyte, isolate two natural products osajin (OSJ) and pomiferin (PMF) from Maclura pomifera fruits, and evaluate the in vitro effect of these natural metabolites on these isoenzymes. These natural products may be used as starting points for drug discovery (like drugs used in several therapeutic applications, including antiglaucoma activity). For the purification procedure, the Sepharose-4B-l-tyrosine-sulphonamide affinity chromatography was used...
March 24, 2017: Archives of Physiology and Biochemistry
https://www.readbyqxmd.com/read/28335327/targeting-at-the-nanoscale-a-novel-s-layer-fusion-protein-enabling-controlled-immobilization-of-biotinylated-molecules
#9
Melinda Varga
With the aim of constructing an S-layer fusion protein that combines both excellent self-assembly and specific ligand i.e., biotin binding ability, streptavidin (aa 16-133) was fused to the S-layer protein of Sporosarcina ureae ATCC 13881 (SslA) devoid of its N-terminal 341 and C-terminal 172 amino acids. The genetically engineered chimeric protein could be successfully produced in E. coli, isolated, and purified via Ni affinity chromatography. In vitro recrystallisation experiments performed with the purified chimeric protein in solution and on a silicon wafer have demonstrated that fusion of the streptavidin domain does not interfere with the self-assembling properties of the S-layer part...
November 4, 2016: Nanomaterials
https://www.readbyqxmd.com/read/28334649/binding-of-tem-1-beta-lactamase-to-beta-lactam-antibiotics-by-frontal-affinity-chromatography
#10
Xiu Chen, Yuhua Li, Yan Zhang, Jianting Yang, Liujiao Bian
TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions...
March 18, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28330749/identification-purification-and-expression-patterns-of-chitinase-from-psychrotolerant-pedobacter-sp-pr-m6-and-antifungal-activity-in%C3%A2-vitro
#11
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/28330257/generation-of-an-inducible-system-to-express-polo-like-kinase-cdc5-as-tap-fusion-protein-during-meiosis-in-saccharomyces-cerevisiae
#12
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
#13
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
#14
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/28328315/analysis-of-protein-target-interactions-of-synthetic-mixtures-by-affinity-lc-ms
#15
Prachi Singh, Kalaipriya Madhaiyan, Minh-Dao Duong-Thi, Brian W Dymock, Sten Ohlson
Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90)...
April 2017: SLAS Discov
https://www.readbyqxmd.com/read/28328210/reevaluating-the-significance-of-estrone-as-an-environmental-estrogen
#16
Gerald T Ankley, David Feifarek, Brett Blackwell, Jenna E Cavallin, Kathleen M Jensen, Michael D Kahl, Shane Poole, Eric Randolph, Travis Saari, Daniel L Villeneuve
Studies worldwide have demonstrated the occurrence of feminized male fish at sites impacted by human and animal wastes. A variety of chemicals could contribute to this phenomenon, but those receiving the greatest attention in terms of research and monitoring have been 17β-estradiol (β-E2) and 17α-ethinylestradiol, due both to their prevalence in the environment and strong estrogenic potency. A third steroid, estrone (E1), also can occur at high concentrations in surface waters, but generally has been of lesser concern due to its relatively lower affinity for vertebrate estrogen receptors...
March 22, 2017: Environmental Science & Technology
https://www.readbyqxmd.com/read/28326614/recombinant-expression-of-porcine-spermadhesin-awn-and-its-phospholipid-interaction-indication-for-a-novel-lipid-binding-property
#17
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/28326061/come-an-essential-response-regulator-negatively-regulates-the-expression-of-the-capsular-polysaccharide-locus-and-attenuates-the-bacterial-virulence-in-streptococcus-pneumoniae
#18
Yuqiang Zheng, Xuemei Zhang, Xiaofang Wang, Libin Wang, Jinghui Zhang, Yibing Yin
The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA)...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28325490/development-and-application-of-a-new-in-line-coupling-of-a-miniaturized-boronate-affinity-monolithic-column-with-capillary-zone-electrophoresis-for-the-selective-enrichment-and-analysis-of-cis-diol-containing-compounds
#19
Maria Betzabeth Espina-Benitez, Jérôme Randon, Claire Demesmay, Vincent Dugas
An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-μm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time...
March 9, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28321886/-inside-out-pegylation-of-bovine-%C3%AE-cross-linked-hemoglobin
#20
Kyle D Webster, Dana Dahhan, Abigail M Otto, Cheyanne L Frosti, William L Dean, Jonathan B Chaires, Kenneth W Olsen
The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to β-cross-linked (βXL-Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species...
March 20, 2017: Artificial Organs
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