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Extraction and purification of membrane proteins

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https://www.readbyqxmd.com/read/28543736/a-rapid-expression-and-purification-condition-screening-protocol-for-membrane-protein-structural-biology
#1
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/28518108/identification-of-plant-ice-binding-proteins-through-assessment-of-ice-recrystallization-inhibition-and-isolation-using-ice-affinity-purification
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
https://www.readbyqxmd.com/read/28065317/preparation-of-centralspindlin-as-an-active-heterotetramer-of-kinesin-and-gtpase-activating-protein-subunits-for-in-vitro-structural-and-functional-assays
#11
M Mishima
Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/27847172/novel-systematic-detergent-screening-method-for-membrane-proteins-solubilization
#12
Elodie Desuzinges Mandon, Morgane Agez, Rebecca Pellegrin, Sébastien Igonet, Anass Jawhari
Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once...
January 15, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/27813588/purification-biochemical-characterization-and-antimicrobial-activity-of-a-new-lipid-transfer-protein-from-coffea-canephora-seeds
#13
G C V Bard, U Zottich, T A M Souza, S F F Ribeiro, G B Dias, S Pireda, M Da Cunha, R Rodrigues, L S Pereira, O L T Machado, A O Carvalho, V M Gomes
Coffee, an agronomical crop of great economic importance, is also among the most commonly traded commodities in worldwide markets. Antimicrobial peptides, which play a role in plant defense, have been identified and isolated particularly from seeds. We isolated and immunolocalized Cc-LTP2, a new lipid transfer protein (LTP) from Coffea canephora seeds. We report its antimicrobial activity against various phytopathogenic fungi of economic importance, and against the bacterium Xanthomonas euvesicatoria. Peptides from C...
October 24, 2016: Genetics and Molecular Research: GMR
https://www.readbyqxmd.com/read/27807400/aqueous-two-phase-system-atps-an-overview-and-advances-in-its-applications
#14
REVIEW
Mujahid Iqbal, Yanfei Tao, Shuyu Xie, Yufei Zhu, Dongmei Chen, Xu Wang, Lingli Huang, Dapeng Peng, Adeel Sattar, Muhammad Abu Bakr Shabbir, Hafiz Iftikhar Hussain, Saeed Ahmed, Zonghui Yuan
Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes...
2016: Biological Procedures Online
https://www.readbyqxmd.com/read/27790201/changes-in-serological-immunology-measures-in-uk-and-kenyan-adults-post-controlled-human-malaria-infection
#15
Susanne H Hodgson, David Llewellyn, Sarah E Silk, Kathryn H Milne, Sean C Elias, Kazutoyo Miura, Gathoni Kamuyu, Elizabeth A Juma, Charles Magiri, Alfred Muia, Jing Jin, Alexandra J Spencer, Rhea J Longley, Thomas Mercier, Laurent Decosterd, Carole A Long, Faith H Osier, Stephen L Hoffman, Bernhards Ogutu, Adrian V S Hill, Kevin Marsh, Simon J Draper
Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27760404/an-improved-non-denaturing-method-for-the-purification-of-spiralin-the-main-membrane-lipoprotein-of-the-pathogenic-bacteria-spiroplasma-melliferum
#16
Yann Desfougères, Jean-Michel Poitou, Henri Wróblewski, Laure Béven
Spiralin is the most abundant protein of several species of spiroplasmas, helical, motile bacteria pathogenic for arthropods and plants. This amphiphilic protein is anchored to the outer face of the plasma membrane by a lipoylated N-terminal cysteine. Although spiroplasma pathogenicity in mammals is controversial, it was shown that spiralin is highly immunogenic and endowed with immunomodulatory activity. In this paper, we describe a high performance method for the purification of Spiroplasma melliferum spiralin under non-denaturing conditions...
November 15, 2016: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/27738959/in-vitro-kinetics-of-p700-reduction-of-thermosynechococcus-elongatus-trimeric-photosystem-i-complexes-by-recombinant-cytochrome-c-6-using-a-joliot-type-led-spectrophotometer
#17
Khoa Nguyen, Michael Vaughn, Paul Frymier, Barry D Bruce
The reduction rate of photo-oxidized Photosystem I (PSI) with various natural and artificial electron donors have been well studied by transient absorption spectroscopy. The electron transfer rate from various donors to P700(+) has been measured for a wide range of photosynthetic organisms encompassing cyanobacteria, algae, and plants. PSI can be a limiting component due to tedious extraction and purification methods required for this membrane protein. In this report, we have determined the in vivo, intracellular cytochrome c 6 (cyt c 6)/PSI ratio in Thermosynechococcus elongatus (T...
January 2017: Photosynthesis Research
https://www.readbyqxmd.com/read/27735901/-creatine-kinase-isoform-b-distribution-in-the-brain-in-schizophrenia
#18
O K Savushkina, E B Tereshkina, T A Prokhorova, E A Vorob'eva, I S Boksha, G Sh Burbaeva
AIM: To compare patterns of brain isoform creatine phosphokinase (CPK B) distributions in post-mortem brain from patients with schizophrenia (Sch) and patients with somatic diseases (controls). MATERIAL AND METHODS: Extracts of readily soluble and membrane-associated proteins were prepared from post-mortem samples of prefrontal cortex (Brodmann area 10), anterior (area 24) and posterior (area 23) cingulate cortex, hippocampus and cerebellum cortex from patients with Sch and control group (the samples were matched by age and postmortem interval)...
2016: Zhurnal Nevrologii i Psikhiatrii Imeni S.S. Korsakova
https://www.readbyqxmd.com/read/27730612/isolation-of-plasmodesmata
#19
Christine Faulkner, Emmanuelle M F Bayer
Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27730565/strategies-for-the-purification-of-membrane-proteins
#20
Sinéad M Smith
Although membrane proteins account for approximately 30 % of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating noncovalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions...
2017: Methods in Molecular Biology
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