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membrane protein purification

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https://www.readbyqxmd.com/read/29143175/vectors-for-expression-of-signal-peptide-dependent-proteins-in-baculovirus-insect-cell-systems-and-their-application-to-expression-and-purification-of-the-high-affinity-immunoglobulin-gamma-fc-receptor-i-in-complex-with-its-gamma-chain
#1
Le T M Le, Jens R Nyengaard, Monika M Golas, Bjoern Sander
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER)...
November 15, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29138064/bicontinuous-microemulsions-as-a-biomembrane-mimetic-system-for-melittin
#2
Douglas G Hayes, Ran Ye, Rachel N Dunlap, Divina B Anunciado, Sai Venkatesh Pingali, Hugh M O'Neill, Volker S Urban
Antimicrobial peptides effectively kill antibiotic-resistant bacteria by forming pores in prokaryotes' biomembranes via penetration into the biomembranes' interior. Bicontinuous microemulsions, consisting of interdispersed oil and water nanodomains separated by flexible surfactant monolayers, are potentially valuable for hosting membrane-associated peptides and proteins due to their thermodynamic stability, optical transparency, low viscosity, and high interfacial area. Here, we show that bicontinuous microemulsions formed by negatively-charged surfactants are a robust biomembrane mimetic system for the antimicrobial peptide melittin...
November 11, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29111637/synthesis-of-cellulose-graft-polypropionic-acid-nanofiber-cation-exchange-membrane-adsorbers-for-high-efficiency-separations
#3
Sahadevan Rajesh, Steven Schneiderman, Caitlin Crandall, Hao Fong, Todd J Menkhaus
Fabrication of membrane adsorbers with elevated binding capacity and high throughput is highly desired for simplifying and improving purification efficiencies of bioproducts (biotherapeutics, vaccines, etc.) in the biotechnological and biopharmaceutical industries. Here we demonstrate the preparation of a novel class of self-supported, cellulose-graft-polypropionic acid (CL-g-PPA) cation-exchange nanofiber membrane adsorbers under mild reaction conditions for the purification of positively charged therapeutic proteins...
November 13, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/29097258/site-specific-o-glycosylation-of-n-terminal-serine-residues-by-polypeptide-galnac-transferase-2-modulates-human-%C3%AE-opioid-receptor-turnover-at-the-plasma-membrane
#4
Jarkko J Lackman, Christoffer K Goth, Adnan Halim, Sergey Y Vakhrushev, Henrik Clausen, Ulla E Petäjä-Repo
G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation...
October 31, 2017: Cellular Signalling
https://www.readbyqxmd.com/read/29090927/purification-and-identification-of-membrane-proteins-from-urinary-extracellular-vesicles-using-triton-x-114-phase-partitioning
#5
Shuiwang Hu, Luca Musante, Dorota Tataruch, Xiaomeng Xu, Oliver Kretz, Michael Henry, Paula Meleady, Haihua Luo, Hequn Zou, Yong Jiang, Harry Holthofer
Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport and signaling. Hence membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall Protein (THP), the most abundant interfering protein in urine...
November 1, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/29081510/isolation-and-characterization-of-urinary-extracellular-vesicles-implications-for-biomarker-discovery
#6
REVIEW
Michael L Merchant, Ilse M Rood, Jeroen K J Deegens, Jon B Klein
Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies)...
December 2017: Nature Reviews. Nephrology
https://www.readbyqxmd.com/read/29067565/simple-mild-one-step-labelling-of-proteins-with-gallium-68-using-a-tris-hydroxypyridinone-bifunctional-chelator-a-68-ga-thp-scfv-targeting-the-prostate-specific-membrane-antigen
#7
Saima Nawaz, Gregory E D Mullen, Kavitha Sunassee, Jayanta Bordoloi, Philip J Blower, James R Ballinger
BACKGROUND: Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa)...
October 25, 2017: EJNMMI Research
https://www.readbyqxmd.com/read/29066155/efficient-solubilization-and-purification-of-highly-insoluble-membrane-proteins-expressed-as-inclusion-bodies-using-perfluorooctanoic-acid
#8
Sarah M Plucinsky, Kyle T Root, Kerney Jebrell Glover
The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample...
October 21, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29043672/preparation-of-highly-enriched-er-membranes-using-free-flow-electrophoresis
#9
Harriet T Parsons
Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29037914/antimicrobial-activity-and-preliminary-mode-of-action-of-plnef-expressed-in-escherichia-coli-against-staphylococci
#10
Xuan Tang, Shuangshuang Wu, Xiaofeng Wang, Qing Gu, Ping Li
Strains belonging to the genus of Staphylococci, such as Staphylococcus aureus are common pathologic bacteria which may cause nosocomial cross infection and food contamination. Plantaricin EF (PlnEF), a two-peptide nonlantibiotic bacteriocin produced by Lactobacillus plantarum strains, shows great inhibitory effects against Gram-positive Staphylococci strains. To overproduce this two-peptide bacteriocin, plnE and plnF genes were cloned into pET32a (+) vector and heterologously expressed in Escherichia coli by fusion with His6-tag in this study...
October 14, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29032467/a-helicobacter-pylori-vacuolating-cytotoxin-a-mouse-dhfr-fusion-protein-triggers-dye-release-from-liposomes
#11
Aung Khine Linn, Nitchakan Samainukul, Somsri Sakdee, Chanan Angsuthanasombat, Gerd Katzenmeier
The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E...
October 14, 2017: Current Microbiology
https://www.readbyqxmd.com/read/29030944/noninvasive-detection-of-nasopharyngeal-carcinoma-based-on-saliva-proteins-using-surface-enhanced-raman-spectroscopy
#12
Xueliang Lin, Duo Lin, Xiaosong Ge, Sufang Qiu, Shangyuan Feng, Rong Chen
The present study evaluated the capability of saliva analysis combining membrane protein purification with surface-enhanced Raman spectroscopy (SERS) for noninvasive detection of nasopharyngeal carcinoma (NPC). A rapid and convenient protein purification method based on cellulose acetate membrane was developed. A total of 659 high-quality SERS spectra were acquired from purified proteins extracted from the saliva samples of 170 patients with pathologically confirmed NPC and 71 healthy volunteers. Spectral analysis of those saliva protein SERS spectra revealed specific changes in some biochemical compositions, which were possibly associated with NPC transformation...
October 2017: Journal of Biomedical Optics
https://www.readbyqxmd.com/read/29030733/high-yield-preparation-of-functionally-active-catalytic-translocation-domain-module-of-botulinum-neurotoxin-type-a-that-exhibits-uniquely-different-enzyme-kinetics
#13
Harkiranpreet Kaur Dhaliwal, Nagarajan Thiruvanakarasu, Raj Kumar, Kruti Patel, Ghuncha Ambrin, Shouwei Cai, Bal Ram Singh
Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively...
October 13, 2017: Protein Journal
https://www.readbyqxmd.com/read/29028485/a-study-on-%C3%AE-glucan-binding-protein-%C3%AE-gbp-and-its-involvement-in-phenoloxidase-cascade-in-indian-white-shrimp-fenneropenaeus-indicus
#14
Mahalingam Anjugam, Baskaralingam Vaseeharan, Arokiadhas Iswarya, Muthu Amala, Marimuthu Govindarajan, Naiyf S Alharbi, Shine Kadaikunnan, Jamal M Khaled, Giovanni Benelli
The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3...
October 10, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/29027450/-cloning-and-expression-of-scavenger-receptor-class-b-bmscrb8-in-silkworm-bombyx-mori
#15
Yuzu Zhao, Kui Zhang, Mei Tang, Man Xu, Chongyang Li, Guangzhao Pan, Li Shen, Hongjuan Cui, Liqun Yang
Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/29026495/recombinant-production-and-affinity-purification-of-the-frac-pore-forming-toxin-using-hexa-his-tag-and-pet-expression-cassette
#16
Mehdi Imani, Hossein Zarei Jaliani, Mohammad Hassan Kheirandish, Mahnaz Azadpour
OBJECTIVES: A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification. MATERIALS AND METHODS: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+)...
April 2017: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/29023592/polymer-based-precipitation-preserves-biological-activities-of-extracellular-vesicles-from-an-endometrial-cell-line
#17
Ziru Niu, Ronald T K Pang, Weimin Liu, Qian Li, Ranran Cheng, William S B Yeung
Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF)...
2017: PloS One
https://www.readbyqxmd.com/read/29019009/electroinduced-extraction-of-human-ferritin-heavy-chain-expressed-in-hansenula-polymorpha
#18
Valentina Ganeva, Bojidar Galutzov, Boyana Angelova, Manfred Suckow
А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H...
October 11, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28994773/improved-method-for-the-establishment-of-an-in-vitro-blood-brain-barrier-model-based-on-porcine-brain-endothelial-cells
#19
Simone S E Nielsen, Piotr Siupka, Ana Georgian, Jane E Preston, Andrea E Tóth, Siti R Yusof, N Joan Abbott, Morten S Nielsen
The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation...
September 24, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28994704/characterization-of-mucosal-disaccharidases-from-human-intestine
#20
Mahdi Amiri, Hassan Y Naim
In this study, we used a brush border membrane (BBM) preparation from human small intestine to analyze the proportion and the activity of major intestinal disaccharidases, including sucrase-isomaltase (SI), maltase-glucoamylase (MGAM) and lactase-phlorizin hydrolase (LPH). SI, MGAM and LPH respectively constituted 8.2%, 2.7% and 1.4% of total BBM protein. The activity of SI and LPH decreased threefold after purification from the brush border membrane, which highlights the effect of membrane microdomains on the functional capacity of these enzymes...
October 10, 2017: Nutrients
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