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membrane protein purification

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https://www.readbyqxmd.com/read/28641610/-biological-characteristics-of-microvesicles-secreted-by-human-peripheral-blood-hematopoietic-stem-cells
#1
Chen Liang, Jun-Hui Wang, Lei Deng, Lu Wang, Yi Wang, Ya-Jing Huang, Tie-Qiang Liu, Bo Cai, Hong-Li Zuo, Qi-Yun Sun, Jian-Hui Qiao, Chang-Lin Yu, Kai-Xun Hu, Hui-Sheng Ai, Mei Guo
OBJECTIVE: To investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis. METHODS: PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay...
June 2017: Zhongguo Shi Yan Xue Ye Xue za Zhi
https://www.readbyqxmd.com/read/28634114/peanut-digestome-identification-of-digestion-resistant-ige-binding-peptides
#2
Luigia Di Stasio, Gianluca Picariello, Mariantonietta Mongiello, Rita Nocerino, Roberto Berni Canani, Simona Bavaro, Linda Monaci, Pasquale Ferranti, Gianfranco Mamone
Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases...
June 17, 2017: Food and Chemical Toxicology
https://www.readbyqxmd.com/read/28631792/nanoscopic-dynamics-of-bicontinous-microemulsions-effect-of-membrane-associated-protein
#3
V K Sharma, Douglas G Hayes, Volker S Urban, Hugh M O'Neill, M Tyagi, E Mamontov
Bicontinous microemulsions (BμE) generally consist of nanodomains formed by surfactant in a mixture of water and oil at nearly equal proportions and are potential candidates for the solubilization and purification of membrane proteins. Here we present the first time report of nanoscopic dynamics of surfactant monolayers within BμEs formed by the anionic surfactant sodium dodecyl sulfate (SDS) measured on the nanosecond to picosecond time scale using quasielastic neutron scattering (QENS). BμEs investigated herein consisted of middle phases isolated from Winsor-III microemulsion systems that were formed by mixing aqueous and oil solutions under optimal conditions...
June 20, 2017: Soft Matter
https://www.readbyqxmd.com/read/28631300/a-bacterial-chloroform-reductive-dehalogenase-purification-and-biochemical-characterization
#4
Bat-Erdene Jugder, Susanne Bohl, Helene Lebhar, Robert D Healey, Mike Manefield, Christopher P Marquis, Matthew Lee
We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF...
June 20, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28627567/dissecting-binding-of-a-%C3%AE-barrel-membrane-protein-by-phage-display
#5
Luz M Meneghini, Sarvind Tripathi, Marcus A Woodworth, Sudipta Majumdar, Thomas L Poulos, Gregory A Weiss
Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal...
June 19, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28627360/the-plasmodium-falciparum-exported-protein-pf3d7_0402000-binds-to-erythrocyte-ankyrin-and-band-4-1
#6
Bikash Shakya, Wesley D Penn, Ernesto S Nakayasu, Douglas J LaCount
Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs...
June 13, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28615092/-purification-of-the-recombinant-com1-and-adaa-of-coxiella-burnetii-and-identification-of-the-antigenicity
#7
Jingxian Liu, Yihong Ji, Zhiyang Shi, Yongjun Jiao
Objective To express and purify two kinds of antigens of Coxiella burnetii (C. burnetii), the main outer membrane protein Com1 and the acute disease antigen A (adaA), in prokaryotic expression system and to validate the two recombinant antigens by mass spectrometry and identify their antigenicity. Methods The gene sequences encoding Com1 and adaA were separately synthesized and constructed into the prokaryotic expression vector pET-20b(+). The constructed vectors were transformed into E.coli BL21(DE3), and the recombinant proteins were induced by IPTG...
June 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28609478/purification-of-family-b-g-protein-coupled-receptors-using-nanodiscs-application-to-human-glucagon-like-peptide-1-receptor
#8
Yingying Cai, Yuting Liu, Kelly J Culhane, Brian T DeVree, Yang Yang, Roger K Sunahara, Elsa C Y Yan
Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e...
2017: PloS One
https://www.readbyqxmd.com/read/28607052/efficient-ultra-high-affinity-chromatography-in-a-one-step-purification-of-complex-proteins
#9
Marina N Vassylyeva, Sergiy Klyuyev, Alexey D Vassylyev, Hunter Wesson, Zhuo Zhang, Matthew B Renfrow, Hengbin Wang, N Patrick Higgins, Louise T Chow, Dmitry G Vassylyev
Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10(-14)-10(-17) M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7)...
June 12, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28589281/preparation-and-characterization-of-glass-hollow-fiber-membrane-for-water-purification-applications
#10
Siti Nurfatin Nadhirah Mohd Makhtar, Mukhlis A Rahman, Ahmad Fauzi Ismail, Mohd Hafiz Dzarfan Othman, Juhana Jaafar
This work discusses the preparation and characterizations of glass hollow fiber membranes prepared using zeolite-5A as a starting material. Zeolite was formed into a hollow fiber configuration using the phase inversion technique. It was later sintered at high temperatures to burn off organic materials and change the zeolite into glass membrane. A preliminary study, that used thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Fourier transform infrared (FTIR), confirmed that zeolite used in this study changed to glass at temperatures above 1000 °C...
June 6, 2017: Environmental Science and Pollution Research International
https://www.readbyqxmd.com/read/28581294/contrast-matched-isotropic-bicelles-a-versatile-tool-to-specifically-probe-the-solution-structure-of-peripheral-membrane-proteins-using-sans
#11
Raphael Dos Santos Morais, Olivier Delalande, Javier Pérez, Liza Mouret, Arnaud Bondon, Anne Martel, Marie-Sousai Appavou, Elisabeth Le Rumeur, Jean-François Hubert, Sophie Combet
Obtaining structural information on integral or peripheral membrane proteins is currently arduous due to the difficulty of their solubilization, purification, and crystallization (for X-ray crystallography (XRC) application). To overcome this challenge, bicelles are known to be a versatile tool for high-resolution structure determination, especially when using solution and/or solid state nuclear magnetic resonance (NMR) and, to a lesser extent, XRC. For proteins not compatible with these high-resolution methods, small-angle X-ray and neutron scattering (SAXS and SANS, respectively) are powerful alternatives to obtain structural information directly in solution...
June 16, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
https://www.readbyqxmd.com/read/28580922/phosphate-binding-protein-from-polaromonas-js666-purification-characterization-crystallization-and-sulfur-sad-phasing
#12
Vanessa R Pegos, Louis Hey, Jacob LaMirande, Rachel Pfeffer, Rosalie Lipsh, Moshe Amitay, Daniel Gonzalez, Mikael Elias
Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs...
June 1, 2017: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/28575937/a-facile-way-to-prepare-anti-fouling-and-blood-compatible-polyethersulfone-membrane-via-blending-with-heparin-mimicking-polyurethanes
#13
Chen Wang, Rui Wang, Yuan Xu, Man Zhang, Fan Yang, Shudong Sun, Changsheng Zhao
In this study, new kinds of heparin-mimicking polyurethanes (PUs) were fabricated conveniently in the mixed solution of dimethyl sulfoxide (DMSO) and water, reducing the usage of organic solvent. Functional groups of SO3H and/or COOH were introduced into PUs with various ratios of SO3H to COOH. The PUs were then blended with polyethersulfone (PES) to fabricate heparin-mimicking modified PES membranes by a phase inversion technique. Then, the microstructures, zeta potentials, water contact angles (WCA) and protein adsorptions of the membranes were characterized...
September 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/28567642/purification-of-plant-receptor-kinases-from-plant-plasma-membranes
#14
Jin Suk Lee
Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28560423/prokaryotic-expression-purification-and-characterization-of-human-cyclooxygenase-2
#15
Xiangzhi Liao, Wenhan Wang, Chuanxi Fan, Ning Yang, Jialiang Zhao, Ying Zhang, Ruijuan Gao, Guannan Shen, Simin Xia, Guiying Li
Cyclooxygenase-2 (COX-2) is a key enzyme which catalyzes the conversion of arachidonic acid (AA) into prostaglandins (PGs). It plays an important role in pathophysiological processes, such as tumorigenesis, angiogenesis, inflammation and tumor cell drug resistance. Therefore, COX-2 has been viewed as an important target for cancer therapy. The preparation of COX-2 protein is an important initial step for the subsequent development of COX-2 inhibitors. In this study, we report a strategy to heterologously express truncated human COX-2 (trCOX-2) in Escherichia coli (E...
May 31, 2017: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/28556541/development-of-a-3-step-straight-through-purification-strategy-combining-membrane-adsorbers-and-resins
#16
Michael D Hughson, Thayana A Cruz, Rimenys J Carvalho, Leda R Castilho
The pressures to efficiently produce complex biopharmaceuticals at reduced costs are driving the development of novel techniques, such as in downstream processing with straight-through processing (STP). This method involves directly and sequentially purifying a particular target with minimal holding steps. This work developed and compared 6 different 3-step STP strategies, combining membrane adsorbers, monoliths and resins, to purify a large, complex and labile glycoprotein from Chinese hamster ovary (CHO) cell culture supernatant...
May 27, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28550165/the-clasp2-protein-interaction-network-in-adipocytes-links-clip2-to-agap3-clasp2-to-g2l1-mark2-and-soga1-and-identifies-soga1-as-a-microtubule-associated-protein
#17
Rikke Kruse, James Krantz, Natalie Barker, Richard Coletta, Ruslan Rafikov, Moulun Luo, Kurt Hoejlund, Lawrence J Mandarino, Paul R Langlais
CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT)...
May 26, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28543736/a-rapid-expression-and-purification-condition-screening-protocol-for-membrane-protein-structural-biology
#18
Dan Sjöstrand, Riccardo Diamanti, Camilla A K Lundgren, Benjamin Wiseman, Martin Högbom
Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents...
May 20, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28542437/cp5-system-for-simple-and-highly-efficient-protein-purification-with-a-c-terminal-designed-mini-tag
#19
Hiroyuki Takeda, Wei Zhou, Kohki Kido, Ryoji Suno, Takahiro Iwasaki, Takuya Kobayashi, Tatsuya Sawasaki
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence)...
2017: PloS One
https://www.readbyqxmd.com/read/28534479/efficient-protein-production-inspired-by-how-spiders-make-silk
#20
Nina Kronqvist, Médoune Sarr, Anton Lindqvist, Kerstin Nordling, Martins Otikovs, Luca Venturi, Barbara Pioselli, Pasi Purhonen, Michael Landreh, Henrik Biverstål, Zigmantas Toleikis, Lisa Sjöberg, Carol V Robinson, Nicola Pelizzi, Hans Jörnvall, Hans Hebert, Kristaps Jaudzems, Tore Curstedt, Anna Rising, Jan Johansson
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT...
May 23, 2017: Nature Communications
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