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membrane protein purification

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https://www.readbyqxmd.com/read/28218648/expression-purification-and-monitoring-of-conformational-changes-of-hcb2-tmh67h8-in-different-membrane-mimetic-lipid-mixtures-using-circular-dichroism-and-nmr-techniques
#1
Elvis K Tiburu, Jianqin Zhuang, Heidimarie N A Fleischer, Patrick K Arthur, Gordon A Awandare
This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly (15)N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8)...
February 17, 2017: Membranes
https://www.readbyqxmd.com/read/28198449/recombinant-vacuolar-iron-transporter-family-homologue-pfvit-from-human-malaria-causing-plasmodium-falciparum-is-a-fe-2-h-exchanger
#2
Paola Labarbuta, Katie Duckett, Catherine H Botting, Osama Chahrour, John Malone, John P Dalton, Christopher J Law
Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe(2+) into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells...
February 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28174683/post-translational-modifications-of-annexin-a2-are-linked-to-its-association-with-perinuclear-nonpolysomal-mrnp-complexes
#3
Ingvild Aukrust, Linn Andersen Rosenberg, Mia Madeleine Ankerud, Vibeke Bertelsen, Hanne Hollås, Jaakko Saraste, Ann Kari Grindheim, Anni Vedeler
Various post-translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal- and membrane-associated protein, AnxA2 can function as a trans-acting protein binding to cis-acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25-phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements...
February 2017: FEBS Open Bio
https://www.readbyqxmd.com/read/28168572/expression-purification-and-functionality-of-bioactive-recombinant-human-vascular-endothelial-growth-factor-vegf165-in-e-coli
#4
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/28168175/preparation-and-immunogenicity-of-influenza-virus-like-particles-using-nitrocellulose-membrane-filtration
#5
Young Chan Park, Jae Min Song
PURPOSE: Nitrocellulose membrane-based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment...
January 2017: Clinical and Experimental Vaccine Research
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
#6
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...
February 2, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28145566/optimization-of-a-biotechnological-multiproduct-batch-plant-design-for-the-manufacture-of-four-different-products-a-real-case-scenario
#7
G Sandoval, D Espinoza, N Figueroa, J A Asenjo
In this work a biotechnological multiproduct batch plant that manufactures four different recombinant proteins for human application is described in some detail. This batch plant design is then optimized with regards to the size of equipment using a mixed-integer linear programming (MILP) formulation recently developed by us in order to find a hypothetical new biotechnological batch plant based on the information of real known processes for the production of the four recombinant protein products. The real plant was divided for practical purposes into 2 sub-processes or facilities: a fermentation facility and a purification facility...
February 1, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28126622/purification-and-immunochemical-characterization-of-pla-l-2-the-profilin-from-plantago-lanceolata
#8
Raquel Moya, Virginia Rubio, Juan Mª Beitia, Jerónimo Carnés, M Angeles López-Matas
Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources...
March 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28116781/improved-plasma-membrane-proteome-coverage-with-a-label-free-non-affinity-purified-workflow
#9
Tina Glisovic-Aplenc, Saar Gill, Lynn A Spruce, Ian R Smith, Hossein Fazelinia, Olga Shestova, Hua Ding, Sarah K Tasian, Richard Aplenc, Steven H Seeholzer
The proteins of the cellular plasma membrane perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the plasma membrane proteome has been challenging to isolate and characterize, and is poorly represented in standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. In this study, we employ sucrose gradient ultracentrifugation for the enrichment of the plasma membrane proteome, without chemical labeling and affinity purification, together with GeLCMS and use subsequent bioinformatics tools to select plasma membrane proteins, herein referred to as the surfaceome...
January 23, 2017: Proteomics
https://www.readbyqxmd.com/read/28110389/a-novel-lectin-from-artocarpus-lingnanensis-induces-proliferation-and-th1-th2-cytokine-secretion-through-cd45-signaling-pathway-in-human-t-lymphocytes
#10
Bo Cui, Lu Li, Qiyan Zeng, Faquan Lin, Lijun Yin, Liejun Liao, Min Huang, Jingping Wang
Lectins are carbohydrate-binding proteins and have been used for purification and characterization of glycoproteins. In this study, a novel 58.9-kDa tetrameric lectin from Artocarpus lingnanensis seeds was purified, characterized, and its mitogenic potential was evaluated. The hemagglutination inhibition assay indicated that Artocarpus lingnanensis lectin (ALL) showed specificity toward galactose. ALL was effectively purified in a single-step using affinity chromatography on a galactose-Sepharose column. ALL showed pH optima between 5...
January 21, 2017: Journal of Natural Medicines
https://www.readbyqxmd.com/read/28109855/heterologous-expression-and-characterization-of-plant-taxadiene-5%C3%AE-hydroxylase-cyp725a4-in-escherichia-coli
#11
John Edward Rouck, Bradley Walters Biggs, Amogh Kambalyal, William R Arnold, Marjan De Mey, Parayil Kumaran Ajikumar, Aditi Das
Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis...
January 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28089451/crystallogenesis-of-membrane-proteins-mediated-by-polymer-bounded-lipid-nanodiscs
#12
Jana Broecker, Bryan T Eger, Oliver P Ernst
For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization...
February 7, 2017: Structure
https://www.readbyqxmd.com/read/28089070/steric-exclusion-chromatography-for-purification-of-cell-culture-derived-influenza-a-virus-using-regenerated-cellulose-membranes-and-polyethylene-glycol
#13
Pavel Marichal-Gallardo, Michael M Pieler, Michael W Wolff, Udo Reichl
Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration...
December 29, 2016: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28088451/efficient-sortase-mediated-n-terminal-labeling-of-tev-protease-cleaved-recombinant-proteins
#14
Kwabena Sarpong, Ron Bose
A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein...
March 15, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28087714/dlg5-connects-cell-polarity-and-hippo-signaling-protein-networks-by-linking-par-1-with-mst1-2
#15
Julian Kwan, Anna Sczaniecka, Emad Heidary Arash, Liem Nguyen, Chia-Chun Chen, Srdjana Ratkovic, Olga Klezovitch, Liliana Attisano, Helen McNeill, Andrew Emili, Valeri Vasioukhin
Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation...
December 15, 2016: Genes & Development
https://www.readbyqxmd.com/read/28069171/steric-exclusion-chromatography-for-purification-of-cell-culture-derived-influenza-a-virus-using-regenerated-cellulose-membranes-and-polyethylene-glycol
#16
Pavel Marichal-Gallardo, Michael M Pieler, Michael W Wolff, Udo Reichl
Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration...
February 3, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28067309/the-autophagy-elongation-complex-atg5-12-16l1-positively-regulates-hcv-replication-and-is-required-for-wild-type-membranous-web-formation
#17
Ahmed M Fahmy, Patrick Labonté
Hepatitis C virus (HCV) infection induces intracellular membrane rearrangements, thus forming a membranous web (MW) in which HCV replication and assembly occur. The HCV-induced MW is primarily composed of double membrane vesicles (DMVs) transfused by multi-membrane vesicles. The autophagy machinery has been proposed to participate in the formation of such vesicles. However, no clear evidence has been found linking autophagy to the formation of these DMVs. In this study, we evaluated the role of the autophagy elongation complex (ATG5-12/16L1) in HCV replication and MW formation...
January 9, 2017: Scientific Reports
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
#18
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/28065274/probing-the-activity-of-eukaryotic-rhomboid-proteases-in-vitro
#19
B Cordier, M K Lemberg
Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065273/screening-and-characterization-strategies-for-nanobodies-targeting-membrane-proteins
#20
S Veugelen, M Dewilde, B De Strooper, L Chávez-Gutiérrez
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites)...
2017: Methods in Enzymology
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