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membrane protein purification

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https://www.readbyqxmd.com/read/29325877/dmcatd-a-cathepsin-d-like-peptidase-of-the-hematophagous-insect-dipetalogaster-maxima-hemiptera-reduviidae-purification-bioinformatic-analyses-and-the-significance-of-its-interaction-with-lipophorin-in-the-internalization-by-developing-oocytes
#1
Jimena Leyria, Leonardo L Fruttero, Rodrigo Ligabue-Braun, Marina S Defferrari, Estela L Arrese, José L Soulages, Beatriz P Settembrini, Celia R Carlini, Lilián E Canavoso
DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry...
January 8, 2018: Journal of Insect Physiology
https://www.readbyqxmd.com/read/29318540/identification-of-proteolytic-cleavage-sites-of-epha2-by%C3%A2-membrane-type-1-matrix-metalloproteinase-on-the%C3%A2-surface-of-cancer-cells
#2
Keiji Kikuchi, Hiroko Kozuka-Hata, Masaaki Oyama, Motoharu Seiki, Naohiko Koshikawa
Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification ...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29288279/isolation-and-characterization-from-solar-salterns-of-north-algeria-of-a-haloarchaeon-producing-a-new-halocin
#3
Souhila Mazguene, Mosè Rossi, Marta Gogliettino, Gianna Palmieri, Ennio Cocca, Sara Mirino, Nacera Imadalou-Idres, Said Benallaoua
Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea...
December 29, 2017: Extremophiles: Life Under Extreme Conditions
https://www.readbyqxmd.com/read/29282287/the-17-residue-long-n-terminus-in-huntingtin-controls-step-wise-aggregation-in-solution-and-on-membranes-via-different-mechanisms
#4
Nitin K Pandey, J Mario Isas, Anoop Rawat, Rachel V Lee, Jennifer Langen, Priyatama Pandey, Ralf Langen
Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: (1) unseeded in solution, (2) seeded in solution, and (3) membrane-mediated...
December 27, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29234081/high-flux-water-purification-using-aluminium-hydroxide-hydrate-gels
#5
Ali Malekizadeh, Peer M Schenk
Filtration of aqueous liquids has wide implications, for example for provision of clean drinking water. Nevertheless, many people still lack access to safe water and suffer from preventable water-borne microbial diseases. This study reports a new ultrafiltration-range separation technology using a gelatinous layer of aluminium hydroxide polyhydrate as a secondary membrane on a retaining fabric that enables simple and cost-effective production of filtered water. Properties include at least 4-fold higher flux rates than currently available membranes, pressure-resistance, impenetrability to filtered particles, easy cleaning by backwashing and simple, cost-effective replacement by gel injection...
December 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29231738/affinity-purification-and-single-molecule-analysis-of-integral-membrane-proteins-from-crude-cell-membrane-preparations
#6
Anders Oskar Lundgren, Björn Johansson Fast, Stephan Block, Björn Agnarsson, Erik Reimhult, Anders Gunnarsson, Fredrik Höök
The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analysed for their function with single-molecule microscopy. Expansion of this approach towards a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilisation and reconstitution often cause protein conformational changes and loss of membrane-specific co-factors, which may impair protein function...
December 12, 2017: Nano Letters
https://www.readbyqxmd.com/read/29223161/cell-free-expression-purification-and-characterization-of-the-functional-%C3%AE-2-adrenergic-receptor-for-multianalyte-detection-of-%C3%AE-agonists
#7
Jian Wang, Yuan Liu, Junhua Zhang, Zhengzheng Han, Wei Wang, Yang Liu, Dong Wei, Wei Huang
Large-scale expression of β2-adrenergic receptor (β2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β2-AR in a cell-free system based on Escherichia coli extracts...
November 2017: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29220051/improving-extraction-and-post-purification-concentration-of-membrane-proteins
#8
Hasin Feroz, HyeYoung Kwon, Jing Peng, Hyeonji Oh, Bryan Ferlez, Carol S Baker, John H Golbeck, Guillermo C Bazan, Andrew L Zydney, Manish Kumar
Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins...
December 8, 2017: Analyst
https://www.readbyqxmd.com/read/29218515/screening-and-scale-up-of-glut-transporter-constructs-suitable-for-biochemical-and-structural-studies
#9
Grégory Verdon, Hae Joo Kang, David Drew
Identifying membrane proteins that can be produced and isolated in homogenous form in detergent is a lengthy trial-and-error process that can be facilitated by fluorescence-based screening approaches. We describe (1) the strategy and protocol of cloning by homologous recombination, (2) whole-cell and in-gel fluorescence measurements to estimate GLUT-GFP fusion protein yields, (3) use of size-exclusion chromatography monitored by fluorescence (FSEC) for assessing the homogeneity of the GLUT-GFP fusion proteins, and (4) the protocol for large-scale production and purification of the Bos taurus GLUT5 construct that enabled its crystal structure determination...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29218514/crystallization-and-structural-determination-of-the-human-glucose-transporters-glut1-and-glut3
#10
Dong Deng, Nieng Yan
Overexpression, purification, and crystallization of eukaryotic membrane proteins represent a major challenge for structural biology. In recent years, we have solved the crystal structures of the human glucose transporters GLUT1 in the inward-open conformation at 3.17 Å resolution and GLUT3 in the outward-open and occluded conformations at 2.4 and 1.5 Å resolutions, respectively. Structural elucidation of these transporters in three distinct functional states reveal the molecular basis for the alternating access transport cycle of this prototypal solute carrier family...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29218513/expression-and-purification-of-rat-glucose-transporter-1-in-pichia-pastoris
#11
Raminta Venskutonytė, Karin Elbing, Karin Lindkvist-Petersson
Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29217202/cell-free-production-purification-and-characterization-of-human-mitochondrial-adp-atp-carriers
#12
Aleksandra Woznicka-Misaila, Céline Juillan-Binard, Delphine Baud, Eva Pebay-Peyroula, Stéphanie Ravaud
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor...
December 4, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29215632/assembly-of-phospholipid-nanodiscs-of-controlled-size-for-structural-studies-of-membrane-proteins-by-nmr
#13
Franz Hagn, Mahmoud L Nasr, Gerhard Wagner
Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29213997/cloning-optimization-of-induction-conditions-and-purification-of-mycobacterium-tuberculosis-rv1733c-protein-expressed-in-escherichia-coli
#14
Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph
Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E...
April 2017: Iranian Journal of Microbiology
https://www.readbyqxmd.com/read/29208950/x-ray-diffraction-reveals-the-intrinsic-difference-in-the-physical-properties-of-membrane-and-soluble-proteins
#15
Xavier Robert, Josiane Kassis-Sahyoun, Nicoletta Ceres, Juliette Martin, Michael R Sawaya, Randy J Read, Patrice Gouet, Pierre Falson, Vincent Chaptal
Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization...
December 5, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29203835/purification-and-functional-comparison-of-nine-human-aquaporins-produced-in-saccharomyces-cerevisiae-for-the-purpose-of-biophysical-characterization
#16
Frederik Bühring Bjørkskov, Simon Lyngaa Krabbe, Casper Normann Nurup, Julie Winkel Missel, Mariana Spulber, Julie Bomholt, Karen Molbaek, Claus Helix-Nielsen, Kamil Gotfryd, Pontus Gourdon, Per Amstrup Pedersen
The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets...
December 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29191736/immunogenicity-of-vibrio-cholerae-outer-membrane-vesicles-secreted-at-various-environmental-conditions
#17
Rezaei Adriani, Seyed Latif Mousavi Gargari, Shahram Nazarian, Samaneh Sarvary, Nafiseh Noroozi
Cholera is caused by toxigenic Vibrio cholerae. It is a significant health problem and an important cause of mortality of children in developing countries. Annually, about 5-7 million people are being infected worldwide, leading to death of 100,000 to 120,000. Immunization using the currently available cholera vaccines has been recommended by World Health Organization (WHO) in areas where cholera is endemic or at risk of outbreaks. Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play important roles in virulence and host-pathogen interaction...
November 27, 2017: Vaccine
https://www.readbyqxmd.com/read/29189771/chemical-synthesis-of-membrane-proteins-by-the-removable-backbone-modification-method
#18
Shan Tang, Chao Zuo, Dong-Liang Huang, Xiao-Ying Cai, Long-Hua Zhang, Chang-Lin Tian, Ji-Shen Zheng, Lei Liu
Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29180752/the-e-coli-dicarboxylic-acid-transporters-daua-act-as-a-signal-transducer-by-interacting-with-the-dcta-uptake-system
#19
Eleni Karinou, Paul A Hoskisson, Alexander Strecker, Gottfried Unden, Arnaud Javelle
The Slc26A/SulP family of ions transporter is ubiquitous and widpsread in all kingdon of life. In E. coli, we have demonstrated that the Slc26 protein DauA is a C4-dicarboxilic acids (C4-diC) transporter active at acidic pH. The main C4-diC transporter active at pH7 is DctA and is induced by C4-diC via the DcuS/R two component system. DctA interacts with DcuS, the membrane embedded histidine kinase, to transfers DcuS to the responsive state, i.e. in the absence of DctA, DcuS is permanently "on", but its activity is C4-diC-dependent when in complex with DctA...
November 27, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29178580/a-cell-free-platform-for-rapid-synthesis-and-testing-of-active-oligosaccharyltransferases
#20
Jennifer A Schoborg, Jasmine Hershewe, Jessica C Stark, Weston Kightlinger, James E Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P DeLisa, Michael C Jewett
Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins...
November 27, 2017: Biotechnology and Bioengineering
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