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Extraction and purification of membrane proteins

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https://www.readbyqxmd.com/read/28918196/expression-and-purification-of-human-and-saccharomyces-cerevisiae-equilibrative-nucleoside-transporters
#1
Rebba C Boswell-Casteel, Jennifer M Johnson, Zygy Roe-Žurž, Kelli D Duggan, Hannah Schmitz, Franklin A Hays
Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes...
September 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28913526/new-penta-saccharide-bearing-tripod-amphiphiles-for-membrane-protein-structure-studies
#2
Muhammad Ehsan, Lubna Ghani, Yang Du, Parameswaran Hariharan, Jonas S Mortensen, Orquidea Ribeiro, Hongli Hu, Georgios Skiniotis, Claus J Loland, Lan Guan, Brian K Kobilka, Bernadette Byrne, Pil Seok Chae
Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins...
September 15, 2017: Analyst
https://www.readbyqxmd.com/read/28803731/purification-and-characterization-of-the-colicin-a-immunity-protein-in-detergent-micelles
#3
Ane Metola, Ana M Bouchet, Marian Alonso-Mariño, Tammo Diercks, Lena Mäler, Félix M Goñi, Ana R Viguera
The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation...
August 10, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28800364/expression-optimization-of-a-cell-membrane-penetrating-human-papillomavirus-type-16-therapeutic-vaccine-candidate-in-nicotiana-benthamiana
#4
Romana J R Yanez, Renate Lamprecht, Milaid Granadillo, Brandon Weber, Isis Torrens, Edward P Rybicki, Inga I Hitzeroth
High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors...
2017: PloS One
https://www.readbyqxmd.com/read/28768815/synthesis-and-self-assembly-of-cellulose-microfibrils-from-reconstituted-cellulose-synthase
#5
Sung Hyun Cho, Pallinti Purushotham, Chao Fang, Cassandra Maranas, Sara M Díaz-Moreno, Vincent Bulone, Jochen Zimmer, Manish Kumar, B Tracy Nixon
Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5...
September 2017: Plant Physiology
https://www.readbyqxmd.com/read/28755363/direct-extraction-and-purification-of-recombinant-membrane-proteins-from-pichia-pastoris-protoplasts
#6
Lucie Hartmann, Estelle Metzger, Noémie Ottelard, Renaud Wagner
In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28753489/a-versatile-method-for-fabricating-ion-exchange-hydrogel-nanofibrous-membranes-with-superb-biomolecule-adsorption-and-separation-properties
#7
Huan Lv, Xueqin Wang, Qiuxia Fu, Yang Si, Xia Yin, Xiaoran Li, Gang Sun, Jianyong Yu, Bin Ding
Construction ion-exchange membranes with superb biomolecules adsorption and purification performance plays a greatly important role in the fields of biotechnological and biopharmaceutical industry, yet still remains an extremely challenging. Herein, we in situ synthesized the cis-butenedioic anhydride grafted poly(vinyl alcohol) hydrogel nanofibrous membranes (CBA-g-PVA HNFM) by combining electrospinning technique with the grafting-copolymerization crosslinking. Taking advantages of the large specific surface area which could provide numerous sites available for functional groups and biomolecules binding, highly tortuous and interconnected porous channel for biomolecules transfer, and enhanced mechanical strength, the resultant CBA-g-PVA HNFM exhibited relatively high binding amount of 170mgg(-1), rapid equilibrium time of 8h towards the biomolecule template of lysozyme, and the performance could be tailored by regulating the buffer properties and protein concentrations...
November 15, 2017: Journal of Colloid and Interface Science
https://www.readbyqxmd.com/read/28702797/purification-and-antibacterial-mechanism-of-fish-borne-bacteriocin-and-its-application-in-shrimp-penaeus-vannamei-for-inhibiting-vibrio-parahaemolyticus
#8
Xinran Lv, Jingfang Du, Yu Jie, Bolin Zhang, Fengling Bai, Hongfei Zhao, Jianrong Li
Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h...
August 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28674884/targeted-approach-for-proteomic-analysis-of-a-hidden-membrane-protein
#9
Tania Martins-Marques, Sandra I Anjo, Teresa Ribeiro-Rodrigues, Bruno Manadas, Henrique Girao
Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28567642/purification-of-plant-receptor-kinases-from-plant-plasma-membranes
#10
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/28543736/a-rapid-expression-and-purification-condition-screening-protocol-for-membrane-protein-structural-biology
#11
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...
August 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28518108/identification-of-plant-ice-binding-proteins-through-assessment-of-ice-recrystallization-inhibition-and-isolation-using-ice-affinity-purification
#12
Melissa Bredow, Heather E Tomalty, Virginia K Walker
Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity...
May 5, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28469466/an-improved-2-dimensional-gel-electrophoresis-method-for-resolving-human-erythrocyte-membrane-proteins
#13
Manoj Kumar, Rajendra Singh, Anil Meena, Bhagwan S Patidar, Rajendra Prasad, Sunil K Chhabra, Surendra K Bansal
The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously...
2017: Proteomic Insights
https://www.readbyqxmd.com/read/28413443/molecular-and-biochemical-characterization-of-recombinant-cel12b-cel8c-and-peh28-overexpressed-in-escherichia-coli-and-their-potential-in-biofuel-production
#14
Eman Ibrahim, Kim D Jones, Keith E Taylor, Ebtesam N Hosseney, Patrick L Mills, Jean M Escudero
BACKGROUND: The high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of celB-, celC-encoded cellulases (cel12B and cel8C) and peh-encoded polygalacturonase (peh28) from Pectobacterium carotovorum subsp. carotovorum (Pcc) was carried out in our previous study using Escherichia coli as a host vector. The current study partially characterizes the enzymes' molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28364041/galectin-3-interacts-with-the-cell-surface-glycoprotein-cd146-mcam-muc18-and-induces-secretion-of-metastasis-promoting-cytokines-from-vascular-endothelial-cells
#15
Florent Colomb, Weikun Wang, Deborah Simpson, Mudaser Zafar, Robert Beynon, Jonathan M Rhodes, Lu-Gang Yu
The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines...
May 19, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28303608/resorcinarene-based-facial-glycosides-implication-of-detergent-flexibility-on-membrane-protein-stability
#16
Hazrat Hussain, Yang Du, Elena Tikhonova, Jonas S Mortensen, Orquidea Ribeiro, Claudia Santillan, Manabendra Das, Muhammad Ehsan, Claus J Loland, Lan Guan, Brian K Kobilka, Bernadette Byrne, Pil Seok Chae
As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups...
May 17, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/28293843/analysis-of-ethylene-receptor-interactions-by-co-immunoprecipitation-assays
#17
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/28225803/bacterial-expression-correct-membrane-targeting-and-functional-folding-of-the-hiv-1-membrane-protein-vpu-using-a-periplasmic-signal-peptide
#18
Arpan Deb, William A Johnson, Alexander P Kline, Boston J Scott, Lydia R Meador, Dustin Srinivas, Jose M Martin-Garcia, Katerina Dörner, Chad R Borges, Rajeev Misra, Brenda G Hogue, Petra Fromme, Tsafrir S Mor
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora...
2017: PloS One
https://www.readbyqxmd.com/read/28168572/expression-purification-and-functionality-of-bioactive-recombinant-human-vascular-endothelial-growth-factor-vegf165-in-e-coli
#19
Awatef Taktak-BenAmar, Maram Morjen, Hazem Ben Mabrouk, Rania Abdelmaksoud-Dammak, Mohamed Guerfali, Najla Fourati-Masmoudi, Naziha Marrakchi, Ali Gargouri
Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28161544/purification-and-characterization-of-peptides-from-capsicum-annuum-fruits-which-are-%C3%AE-amylase-inhibitors-and-exhibit-high-antimicrobial-activity-against-fungi-of-agronomic-importance
#20
Layrana de Azevedo Dos Santos, Gabriel Bonan Taveira, Suzanna de Fátima Ferreira Ribeiro, Lídia da Silva Pereira, André de Oliveira Carvalho, Rosana Rodrigues, Antônia Elenir Amâncio Oliveira, Olga Lima Tavares Machado, Jucélia da Silva Araújo, Ilka Maria Vasconcelos, Valdirene Moreira Gomes
Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5...
April 2017: Protein Expression and Purification
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