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membrane protein purification

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https://www.readbyqxmd.com/read/28326614/recombinant-expression-of-porcine-spermadhesin-awn-and-its-phospholipid-interaction-indication-for-a-novel-lipid-binding-property
#1
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/28324719/negatively-charged-polysulfone-membranes-with-hydrophilicity-and-antifouling-properties-based-on-in-situ-cross-linked-polymerization
#2
Lijing Zhu, Haiming Song, Dawei Zhang, Gang Wang, Zhixiang Zeng, Qunji Xue
Polysulfone (PSf) membrane has been widely used in water separation and purification, although, membrane fouling is still a serious problem limiting its potential. We aim to improve the antifouling of PSf membranes via a very simple and efficient method. In this work, antifouling PSf membranes were fabricated via in situ cross-linked polymerization coupled with non-solvent induced phase separation. In brief, acrylic acid (AA) and vinyltriethoxysilane (VTEOS) were copolymerized in PSf solution, then directly casted into membranes without purification...
March 14, 2017: Journal of Colloid and Interface Science
https://www.readbyqxmd.com/read/28315746/optimized-protocol-for-soluble-prokaryotic-expression-purification-and-structural-analysis-of-human-placenta-specific-1-plac1
#3
Mahboobeh Nazari, Amir-Hassan Zarnani, Roya Ghods, Rahman Emamzadeh, Somayeh Najafzadeh, Arash Minai-Tehrani, Jafar Mahmoudian, Maryam Yousefi, Sedigheh Vafaei, Sam Massahi, Mohammad-Reza Nejadmoghaddam
Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles...
March 16, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28303608/resorcinarene-based-facial-glycosides-implication-of-detergent-flexibility-on-membrane-protein-stability
#4
Pil Seok Chae, Hazrat Hussain, Yang Du, Elena Tikhonova, Jonas Mortensen, Orquidea Ribeiro, Claudia Santillan, Manabendra Das, Claus Løland, Lan Guan, Brian Kobilka, Bernadette Byrne, Muhammad Ehsan
As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from the lipid environment and to maintain their solubility and stability in an aqueous medium. Despite the wide utility in membrane protein research, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of novel agents with enhanced properties. In the current study we introduce two classes of novel amphiphiles, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), where the alkyl chains are facially segregated from the carbohydrate head groups...
March 17, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/28302513/expression-purification-and-enzymatic-characterization-of-undecaprenyl-pyrophosphate-phosphatase-from-vibrio-vulnificus
#5
Hsin-Yang Chang, Chia-Cheng Chou, Mao-Lun Wu, Andrew H J Wang
Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E...
March 14, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28299730/expression-and-purification-of-a-matrix-metalloprotease-transmembrane-domain-in-escherichia-coli
#6
Charles A Galea
Membrane tethered matrix metalloproteases are bound to the plasma membrane by a glycosylphosphatidylinositol-anchor or a transmembrane domain. To date, most studies of membrane-bound matrix metalloprotease have focused on the globular catalytic and protein-protein interaction domains of these enzymes. However, the transmembrane domains have been poorly studied even though they are known to mediate intracellular signaling via interaction with various cellular proteins. The expression and purification of the transmembrane domain of these proteins can be challenging due to their hydrophobic nature...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28296951/in-silico-design-expression-and-purification-of-novel-chimeric-escherichia-coli-o157-h7-ompa-fused-to-ltb-protein-in-escherichia-coli
#7
Aytak Novinrooz, Taghi Zahraei Salehi, Roya Firouzi, Sina Arabshahi, Abdollah Derakhshandeh
E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E...
2017: PloS One
https://www.readbyqxmd.com/read/28293843/analysis-of-ethylene-receptor-interactions-by-co-immunoprecipitation-assays
#8
Zhiyong Gao, G Eric Schaller
Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28291804/purification-of-cone-outer-segment-for-proteomic-analysis-on-its-membrane-proteins-in-carp-retina
#9
Takashi Fukagawa, Kazuaki Takafuji, Shuji Tachibanaki, Satoru Kawamura
Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones...
2017: PloS One
https://www.readbyqxmd.com/read/28289087/reoxidation-of-the-thiol-disulfide-oxidoreductase-mdba-by-a-bacterial-vitamin-k-epoxide-reductase-in-the-biofilm-forming-actinobacterium-actinomyces-oris
#10
Truc Thanh Luong, Melissa E Reardon-Robinson, Sara D Siegel, Hung Ton-That
Post-translocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial Vitamin K Epoxide Reductase (VKOR)-like protein that contains four cysteine residues C93/C101 and C175/C178 with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known...
March 13, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28287548/legionella-pneumophila-outer-membrane-vesicles-isolation-and-analysis-of-their-pro-inflammatory-potential-on-macrophages
#11
Anna Lena Jung, Kerstin Hoffmann, Christina E Herkt, Christine Schulz, Wilhelm Bertrams, Bernd Schmeck
Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole...
February 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28283569/the-novel-chloroplast-outer-membrane-kinase-koc1-is-a-required-component-of-the-plastid-protein-import-machinery
#12
Mónica Zufferey, Cyrille Montandon, Véronique Douet, Emilie Demarsy, Birgit Agne, Sacha Baginsky, Felix Kessler
The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts requires the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on Translocons at the Outer and Inner Chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo...
March 10, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28277945/polymer-membrane-ion-selective-electrodes-as-a-convenient-tool-for-lipases-and-esterases-assays
#13
Maciej Cieplak, Ryszard Ostaszewski
We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored...
March 9, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28277548/optimized-labeling-of-membrane-proteins-for-applications-to-super-resolution-imaging-in-confined-cellular-environments-using-monomeric-streptavidin
#14
Ingrid Chamma, Olivier Rossier, Grégory Giannone, Olivier Thoumine, Matthieu Sainlos
Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM))...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28274101/enhanced-purification-of-recombinant-rat-nadph-p450-reductase-by-using-a-hexahistidine-tag
#15
Hyoung-Goo Park, Young-Ran Lim, Songhee Han, Dabin Jeong, Donghak Kim
NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum (ER) membrane and utilizes FMN, FAD, and NADPH as cofactors. While NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP⁺ in the affinity chromatography process...
March 9, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28272458/modulation-of-lat1-slc7a5-transporter-activity-and-stability-by-membrane-cholesterol
#16
David Dickens, George N Chiduza, Gareth S A Wright, Munir Pirmohamed, Svetlana V Antonyuk, S Samar Hasnain
LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA)...
March 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28255712/purification-of-lat-containing-membranes-from-resting-and-activated-t-lymphocytes
#17
Claire Hivroz, Paola Larghi, Mabel Jouve, Laurence Ardouin
In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin(®) resin, allowing the purification of membranes containing the Strep-tagged molecule of interest...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28253967/in-vitro-reconstitution-of-atg8-conjugation-and-deconjugation
#18
D Fracchiolla, B Zens, S Martens
Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28253953/methods-for-studying-interactions-between-atg8-lc3-gabarap-and-lir-containing-proteins
#19
T Johansen, Å B Birgisdottir, J Huber, A Kniss, V Dötsch, V Kirkin, V V Rogov
LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28248007/antibody-purification-from-cho-cell-supernatant-using-new-multimodal-membranes
#20
Juan Wang, Jinxiang Zhou, Yogender K Gowtham, Sarah W Harcum, Scott M Husson
This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG1 following a size-exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two step MMM chromatography process achieved high selectivity for capturing hIgG1 from the CHO cell culture supernatant, though the desalting step resulted in product dilution...
March 1, 2017: Biotechnology Progress
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