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Extraction and purification of membrane proteins

V Vasilca, A Sadeghpour, S Rawson, L E Hawke, S A Baldwin, T Wilkinson, D Bannister, V L G Postis, M Rappolt, S P Muench, L J C Jeuken
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment...
March 12, 2018: Analytical Biochemistry
Farida Larit, Khaled M Elokely, Narayan D Chaurasiya, Samira Benyahia, Manal A Nael, Francisco León, Mohammad Sanad Abu-Darwish, Thomas Efferth, Yan-Hong Wang, Djamila Belouahem-Abed, Samir Benayache, Babu L Tekwani, Stephen J Cutler
BACKGROUND: Monoamine oxidases (MAOs) are outer mitochondrial membrane flavoenzymes. They catalyze the oxidative deamination of a variety of neurotransmitters. MAO-A and MAO-B may be considered as targets for inhibitors to treat neurodegenerative diseases and depression and for managing symptoms associated with Parkinson's and Alzheimer's diseases. PURPOSE: The objective was to evaluate the inhibitory effect of Hypericum afrum and Cytisus villosus against MAO-A and B and to isolate the compounds responsible for the MAO-inhibitory activity...
February 1, 2018: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
Janine Habier, Patrick May, Anna Heintz-Buschart, Anubrata Ghosal, Anke K Wienecke-Baldacchino, Esther N M Nolte-'t Hoen, Paul Wilmes, Joëlle V Fritz
Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans, lipopolysaccharides, DNA, and RNA. To examine if OMV-associated RNA molecules are bacterial degradation products and/or are functionally active, it is necessary to extract RNA from pure OMVs for subsequent analysis. Therefore, we describe here an isolation method of ultrapure OMVs and the subsequent extraction of RNA and basic steps of RNA-Seq analysis...
2018: Methods in Molecular Biology
Ruth Skrzypek, Shagufta Iqbal, Richard Callaghan
Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrates. Reconstitution may take many forms and these have been classified into two broad categories...
February 15, 2018: Methods: a Companion to Methods in Enzymology
Kátia G Carvalho, Felipe H S Bambirra, Jacques R Nicoli, Jamil S Oliveira, Alexandre M C Santos, Marcelo P Bemquerer, Antonio Miranda, Bernadette D G M Franco
Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb...
January 18, 2018: Archives of Microbiology
Jian Wang, Yuan Liu, Junhua Zhang, Zhengzheng Han, Wei Wang, Yang Liu, Dong Wei, Wei Huang
Large-scale expression of β2 -adrenergic receptor (β2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β2 -AR in a cell-free system based on Escherichia coli extracts...
November 2017: Biochemistry. Biokhimii︠a︡
Hasin Feroz, HyeYoung Kwon, Jing Peng, Hyeonji Oh, Bryan Ferlez, Carol S Baker, John H Golbeck, Guillermo C Bazan, Andrew L Zydney, Manish Kumar
Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins...
March 12, 2018: Analyst
Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph
Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis , a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E...
April 2017: Iranian Journal of Microbiology
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
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
Benjamin Ifeoluwa Okeleye, Noxolo Thabiso Mkwetshana, Roland Ndip Ndip
We evaluated the in-vitro antiproliferative and apoptotic potential of the ethyl acetate extract (EAE) of Peltophorum africanum, a member of the family Fabaceae (Sond) in order to validate its pharmacological use. Antiproliferation of human breast (MCF-7), colon (HT-29) and cervical (HeLa) cancer cell lines by EAE was investigated using the Cell Titer-Blue viability assay and the mechanism of action delineated using the Nucleic Acid and Protein Purification Nucleospin(®) Tissue Kit, Scanning Electron Microscope (SEM), propidium iodide (PI) and acridine orange (AO) double-staining techniques...
2017: Iranian Journal of Pharmaceutical Research: IJPR
Rebba C Boswell-Casteel, Jennifer M Johnson, Zygy Roe-Žurž, Kelli D Duggan, Hannah Schmitz, Franklin A Hays
Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes...
February 2018: Protein Expression and Purification
Muhammad Ehsan, Lubna Ghani, Yang Du, Parameswaran Hariharan, Jonas S Mortensen, Orquidea Ribeiro, Hongli Hu, Georgios Skiniotis, Claus J Loland, Lan Guan, Brian K Kobilka, Bernadette Byrne, Pil Seok Chae
Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins...
September 15, 2017: Analyst
Ane Metola, Ana M Bouchet, Marian Alonso-Mariño, Tammo Diercks, Lena Mäler, Félix M Goñi, Ana R Viguera
The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation...
November 2017: Biochimica et Biophysica Acta
Romana J R Yanez, Renate Lamprecht, Milaid Granadillo, Brandon Weber, Isis Torrens, Edward P Rybicki, Inga I Hitzeroth
High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors...
2017: PloS One
Sung Hyun Cho, Pallinti Purushotham, Chao Fang, Cassandra Maranas, Sara M Díaz-Moreno, Vincent Bulone, Jochen Zimmer, Manish Kumar, B Tracy Nixon
Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5...
September 2017: Plant Physiology
Lucie Hartmann, Estelle Metzger, Noémie Ottelard, Renaud Wagner
In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process...
2017: Methods in Molecular Biology
Huan Lv, Xueqin Wang, Qiuxia Fu, Yang Si, Xia Yin, Xiaoran Li, Gang Sun, Jianyong Yu, Bin Ding
Construction ion-exchange membranes with superb biomolecules adsorption and purification performance plays a greatly important role in the fields of biotechnological and biopharmaceutical industry, yet still remains an extremely challenging. Herein, we in situ synthesized the cis-butenedioic anhydride grafted poly(vinyl alcohol) hydrogel nanofibrous membranes (CBA-g-PVA HNFM) by combining electrospinning technique with the grafting-copolymerization crosslinking. Taking advantages of the large specific surface area which could provide numerous sites available for functional groups and biomolecules binding, highly tortuous and interconnected porous channel for biomolecules transfer, and enhanced mechanical strength, the resultant CBA-g-PVA HNFM exhibited relatively high binding amount of 170mgg(-1), rapid equilibrium time of 8h towards the biomolecule template of lysozyme, and the performance could be tailored by regulating the buffer properties and protein concentrations...
November 15, 2017: Journal of Colloid and Interface Science
Xinran Lv, Jingfang Du, Yu Jie, Bolin Zhang, Fengling Bai, Hongfei Zhao, Jianrong Li
Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h...
August 2017: World Journal of Microbiology & Biotechnology
Tania Martins-Marques, Sandra I Anjo, Teresa Ribeiro-Rodrigues, Bruno Manadas, Henrique Girao
Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS...
2017: Methods in Molecular Biology
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