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Extraction and purification of membrane proteins

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...
October 12, 2016: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
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...
October 13, 2016: Photosynthesis Research
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
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
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
Kerrie A Morrison, Aneel Akram, Ashlyn Mathews, Zoeya A Khan, Jaimin H Patel, Chumin Zhou, David John Hardy, Charles Moore-Kelly, Roshani Patel, Victor Odiba, Tim Knowles, Masood-Ul-Hassan Javed, Nikola P Chmel, Timothy R Dafforn, Alice Jane Rothnie
The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins...
September 30, 2016: Biochemical Journal
Pedro F N Souza, Ilka M Vasconcelos, Fredy D A Silva, Frederico B Moreno, Ana C O Monteiro-Moreira, Luciana M R Alencar, Angélica S G Abreu, Jeanlex S Sousa, José T A Oliveira
Hospital-acquired infections caused by antibiotic-resistant bacteria threaten the lives of many citizens all over the world. Discovery of new agents to hinder bacterial development would have a significant impact on the treatment of infections. Here, the purification and characterization of Rc-2S-Alb, a protein that belongs to the 2S albumin family, from Ricinus communis seed cake, are reported. Rc-2S-Alb was purified after protein extraction with Tris-HCl buffer, pH 7.5, fractionation by ammonium sulfate (50-75%), and chromatography on Phenyl-Sepharose and DEAE-Sepharose...
September 28, 2016: Journal of Natural Products
Angelica Tamayo Tenorio, Remko M Boom, Atze Jan van der Goot
Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics. Therefore, this study analysed proteomic extraction methods for membrane proteins as an inspiration for a food-grade alternative process. Sugar beet leaves were extracted with two proteomic protocols: solvent extraction and Triton X-114 phase partitioning method...
February 15, 2017: Food Chemistry
A J M Kouyoumdjian, R A Lazar, N K H Slater
In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12...
October 14, 2016: Journal of Chromatography. A
Debasis Das, Marthe T C Walvoort, Vinita Lukose, Barbara Imperiali
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays...
2016: Scientific Reports
Alexey S Vasilchenko, Eugene A Rogozhin, Anastasia V Vasilchenko, Olga L Kartashova, Maria V Sycheva
THE AIM: of this work was to purify and characterize antimicrobial peptide derived from acid extract of Gallus gallus blood cells. METHODS AND RESULTS: Two polypeptides (i.e., CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (Gallus gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC)...
September 1, 2016: Journal of Applied Microbiology
Jimin Xiong, Danijela Menicanin, Peter S Zilm, Victor Marino, P Mark Bartold, Stan Gronthos
The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells...
2016: Stem Cells International
Anandhi Anandan, Alice Vrielink
Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers...
2016: Advances in Experimental Medicine and Biology
Sarah C Lee, Naomi L Pollock
The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques...
August 15, 2016: Biochemical Society Transactions
Feng-Wei Ma, Si-Yuan Kong, Hong-Sheng Tan, Rong Wu, Bing Xia, Yan Zhou, Hong-Xi Xu
In the present study, a novel polysaccharide, PSP-2B, was isolated from aqueous extracts of Prunellae Spica by direct ultrafiltration membrane separation and gel chromatography purification. PSP-2B is a partially sulphated polysaccharide with a molecular weight of approximately 32kDa. Its sulfate content is 10.59% by elemental analysis. The major sugars comprising PSP-2B are arabinose, galactose and mannose, in addition to small amounts of glucose and uronic acids. The framework of PSP-2B is speculated to be a branched arabinogalactomannan, and the side chains are terminated primarily by the Araf residues...
November 5, 2016: Carbohydrate Polymers
Alice J Rothnie
Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents...
2016: Methods in Molecular Biology
Benjamin Wiseman, Jean-Michel Jault
The production and purification is normally the first step in any biophysical or biochemical study of a new target protein. For membrane proteins, due to their generally low expression levels and hydrophobic properties this is often a major hurdle. Some multidrug transporters are members of one of the largest families of membrane proteins, the ABC ("ATP-binding cassette"), and are responsible for the uptake and export of a wide variety of molecules. This can lead to resistance when those molecules are antibiotics or chemotherapy drugs...
2016: Methods in Molecular Biology
Maria Arvaniti, Majbrit M Jensen, Neeraj Soni, Hong Wang, Anders B Klein, Nathalie Thiriet, Lars H Pinborg, Pretal P Muldoon, Jacob Wienecke, M Imad Damaj, Kristi A Kohlmeier, Marjorie C Gondré-Lewis, Jens D Mikkelsen, Morten S Thomsen
Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross-linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane-bound protein that binds to multiple nAChR subtypes in the human brain...
September 2016: Journal of Neurochemistry
Sarah C Lee, Tim J Knowles, Vincent L G Postis, Mohammed Jamshad, Rosemary A Parslow, Yu-Pin Lin, Adrian Goldman, Pooja Sridhar, Michael Overduin, Stephen P Muench, Timothy R Dafforn
Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems...
July 2016: Nature Protocols
Benjamin Clémençon, Michael Fine, Matthias A Hediger
The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC...
August 2016: Biochimie
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