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membrane protein purification

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https://www.readbyqxmd.com/read/28089451/crystallogenesis-of-membrane-proteins-mediated-by-polymer-bounded-lipid-nanodiscs
#1
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...
January 3, 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
#2
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
#3
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...
January 11, 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
#4
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
#5
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/28067309/the-autophagy-elongation-complex-atg5-12-16l1-positively-regulates-hcv-replication-and-is-required-for-wild-type-membranous-web-formation
#6
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
#7
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
#8
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
#9
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
https://www.readbyqxmd.com/read/28065266/production-of-recombinant-rhomboid-proteases
#10
E Arutyunova, R Panigrahi, K Strisovsky, M J Lemieux
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063492/interfacial-enzymes-membrane-binding-orientation-membrane-insertion-and-activity
#11
S A Tatulian
Most interfacial enzymes undergo activation upon membrane binding. Interfacial activation is determined not only by the binding strength but also by the specific mode of protein-membrane interactions, including the angular orientation and membrane insertion of the enzymes. This chapter describes biophysical techniques to quantitatively evaluate membrane binding, orientation, membrane insertion, and activity of secreted phospholipase A2 (PLA2) and lipoxygenase (LO) enzymes. Procedures for recombinant production and purification of human pancreatic PLA2 and human 5-lipoxygenase (5-LO) are also presented...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28056928/new-ligation-independent-cloning-vectors-for-expression-of-recombinant-proteins-with-a-self-cleaving-cpd-6xhis-tag
#12
Marco Biancucci, Jazel S Dolores, Jennifer Wong, Sarah Grimshaw, Wayne F Anderson, Karla J F Satchell, Keehwan Kwon
BACKGROUND: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. RESULTS: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag"...
January 5, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28042837/purification-and-characterization-of-antioxidant-peptides-of-pseudosciaena-crocea-protein-hydrolysates
#13
Ningning Zhang, Chong Zhang, Yuanyuan Chen, Baodong Zheng
Two peptides with antioxidant activity were isolated from Pseudosciaena crocea proteins. Pseudosciaena crocea muscle was hydrolyzed with neutral protease to obtain Pseudosciaena crocea protein hydrolysates (PCPH). After ultrafiltration through molecular weight cut-off membranes of 10, 5 and 3 kDa and assessment of free radical scavenging ability, the fraction (PCPH-IV) with the highest antioxidant activity was obtained. Several purification steps, i.e., ion exchange chromatography, gel filtration chromatography and reversed phase high performance liquid chromatography, were applied to further purify PCPH-IV...
December 30, 2016: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28042595/expression-purification-and-crystallization-of-the-herpesvirus-nuclear-egress-complex-nec
#14
Janna M Bigalke, Ekaterina E Heldwein
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures...
July 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28028176/a-two-component-nox-like-system-in-bacteria-is-involved-in-the-electron-transfer-chain-to-the-methionine-sulfoxide-reductase-msrp
#15
Celine Juillan-Binard, Antoine Picciocchi, Jean-Pierre Andrieu, Jerome Dupuy, Isabelle Petit-Hartlein, Christelle Caux-Thang, Corinne Vivès, Vincent Nivière, Franck Fieschi
MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins. A periplasmic one, MsrP previously named YedY, carrying out the Msr activity and MsrQ, an integral b-type heme membrane spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX)...
December 27, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28025687/characterisation-of-the%C3%A2-daacs%C3%A2-family-escherichia-coli-glutamate-aspartate-proton-symporter-gltp-using-computational-chemical-biochemical-and-biophysical-methods
#16
Moazur Rahman, Fouzia Ismat, Li Jiao, Jocelyn M Baldwin, David J Sharples, Stephen A Baldwin, Simon G Patching
Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh...
December 26, 2016: Journal of Membrane Biology
https://www.readbyqxmd.com/read/28000366/hemocompatiblilty-evaluation-of-poly-1-8-octanediol-citrate-blend-polyethersulfone-membranes
#17
Muhamad Zulhilmi Zailani, Ahmad Fauzi Ismail, Siti Hamimah Sheikh Abdul Kadir, Mohd Hafiz Dzarfan Othman, Pei Sean Goh, Hasrinah Hasbullah, Mohd Sohaimi Abdullah, Be Cheer Ng, Fatmawati Kamal
In this study, poly (1,8- octanediol citrate) (POC) was used to modify polyethersufone (PES) based membrane to enhance its hemocompatibility. Different compositions of POC (0-3%) were added into the polyethersulfone (PES) dope solutions and polyvinylpyrrolidone (PVP) was used as pore forming agent. The hemocompatible POC modified PES membranes were fabricated through phase-inversion technique. The prepared membranes were characterized using attenuated total reflectance-fourier transform infrared (ATR-FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), Atomic-force microscopy (AFM), contact angle, Zeta-potential, membrane porosity and pore size and pure water flux (PWF) and BSA rejection...
December 21, 2016: Journal of Biomedical Materials Research. Part A
https://www.readbyqxmd.com/read/27974566/cd63-regulates-epstein-barr-virus-lmp1-exosomal-packaging-enhancement-of-vesicle-production-and-non-canonical-nf-%C3%AE%C2%BAb-signaling
#18
Stephanie N Hurwitz, Dingani Nkosi, Meghan M Conlon, Sara B York, Xia Liu, Deanna C Tremblay, David G Meckes
: Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles...
December 14, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27951570/biocompatibility-of-hemodiafilters
#19
Tadashi Tomo
BACKGROUND: Biocompatibility and the efficiency of solute removal are important considerations in blood purification therapy. Improvement of biocompatibility is expected to lead to the prevention of dialysis-related complications (e.g. amyloidosis, arteriosclerosis, and malnutrition) and to the delay of disease progression by alleviating microinflammation. SUMMARY: The biocompatibility of dialyzers is greatly influenced by the interaction between blood and the treatment materials, in which the chemical and physical characteristics of membrane materials play important roles...
2017: Contributions to Nephrology
https://www.readbyqxmd.com/read/27951567/evidence-for-targeting-low-molecular-weight-proteins-in-hemodialysis-and-hemodiafiltration
#20
Kenji Tsuchida, Kojiro Nagai, Narushi Yokota, Satoru Yamada, Hiroyuki Michiwaki, Jun Minakuchi
BACKGROUND: With the identification of β2-microglobulin (β2MG) as an active participant in dialysis-related amyloid fibril formation, low-molecular-weight proteins (LMWPs) are now recognized as a distinct class of uremic toxins, and numerous compounds in this category have been identified. The class of LMWPs, although not precisely defined, has a molecular weight range of approximately 1,000-50,000 Da. With this in mind, dialysis prescriptions have been modified to increase the efficiency of uremic solute removal...
2017: Contributions to Nephrology
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