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membrane protein purification

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...
September 13, 2017: 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
Kris Wilson, Scott P Webster, John Peter Iredale, Xiaozhong Zheng, Natalie Z M Homer, Nhan Pham, Manfred Auer, Damian James Mole
The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity - the Toxicity-Affinity-Permeability-Selectivity (TAPS) technique...
September 13, 2017: Methods and Applications in Fluorescence
Charlotte Roth, Daniel Lüdke, Melanie Klenke, Annalena Quathamer, Oliver Valerius, Gerhard H Braus, Marcel Wiermer
Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the auto-immune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6...
September 13, 2017: Plant Journal: for Cell and Molecular Biology
Carissa Pilling, Jonathan A Cooper
Suppressors of cytokine signaling (SOCS) proteins inhibit signaling by serving as substrate receptors for the Cullin5-RING E3 ubiquitin ligase (CRL5) and through a variety of CRL5-independent mechanisms. CRL5, SOCS2 and SOCS6 are implicated in suppressing transformation of epithelial cells. We identified cell proteins that interact with SOCS2 and SOCS6 using two parallel proteomics techniques: BioID and Flag affinity purification mass spectrometry. The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) was identified as a SOCS2-interacting protein...
September 7, 2017: Scientific Reports
Małgorzata Szczygieł, Marcin Markiewicz, Milena Szafraniec, Roxana Zuziak, Krystyna Urbańska, Leszek Fiedor
All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein...
2017: Acta Biochimica Polonica
Chandrika N Deshpande, Vicky Xin, Yan Lu, Tom Savage, Gregory J Anderson, Mika Jormakka
Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling...
2017: PloS One
D Taladrid, D Marín, A Alemán, I Álvarez-Acero, P Montero, M C Gómez-Guillén
The effect of two-step and five-step acetone washing of soybean lecithin (SL) on compositional properties of partially purified phosphatidylcholines (PW2 and PW5) was studied. Trace amounts of protein were detected in SL, PW2 and PW5, with a predominance of glutamic acid and aspartic acid. Increasing the number of acetone washing steps significantly reduced the total content of γ-, δ- and α-tocopherol. Similar reductions (≈90%) of neutral lipids were found in both PW2 and PW5, but the removal of free fatty acids was higher in PW5 than in PW2 (78% vs...
October 2017: Food Research International
Benjamin Stenger, Adrian Gerber, Rita Bernhardt, Frank Hannemann
Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions...
September 1, 2017: Biochimica et Biophysica Acta
Lea Talmann, Jochen Wiesner, Andreas Vilcinskas
Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state...
September 26, 2017: Zeitschrift Für Naturforschung. C, A Journal of Biosciences
Naomi L Pollock, Sarah C Lee, Jaimin H Patel, Aiman A Gulamhussein, Alice J Rothnie
New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer...
August 30, 2017: Biochimica et Biophysica Acta
Qiu-Ping Li, Shuai Wang, Jin-Ying Gou
BACKGROUND: Membrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications. METHODS: First, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus...
September 2, 2017: BMC Biotechnology
King Pong Leung, Wilson Chun Yu Lau
The exocyst, conserved from yeast to plants to mammals, is a hetero-octameric complex that mediates tethering of secretory vesicles to designated sites on the plasma membrane during polarized exocytosis. Because structural studies of the intact exocyst complex have been greatly limited by the low yields of purified proteins, many aspects of the exocyst functions remain poorly understood. Here, we present the protocols for the isolation and purification of the recombinant and the native plant exocyst complex...
2017: Methods in Molecular Biology
Zhao Li, Andrew L Zydney
There is renewed interest in the possibility of using precipitation for initial capture of high value therapeutic proteins as part of an integrated continuous downstream process. These precipitates can be continuously washed using tangential flow filtration, with long term operation achieved by operating the membrane modules below the critical filtrate flux for fouling. Our hypothesis was that the critical flux for the precipitated protein would be a function of the properties of the precipitate as determined by the precipitation conditions...
August 25, 2017: Biotechnology Progress
Richard J Delle Bovi, W Todd Miller
The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization...
August 19, 2017: Analytical Biochemistry
Nagarajan Padmavathy, Paresh Kumar Samantaray, Lopamudra Das Ghosh, Giridhar Madras, Suryasarathi Bose
Polymeric architectures with controlled and well-defined structural features are required to render a sustainable antibacterial surface - a key requirement in the design of polymeric membranes for water purification. Herein, surface selective crosslinking of copper oxide-polyphosphoester (CuO-PPE) hybrid nanogels on to polyvinylidene fluoride-styrene maleic anhydride (PVDF/SMA) ultrafiltration membranes was developed. The hybrid nanogels, composed of PPE and CuO, with inherent antifouling and antibacterial properties, were crosslinked using a macroinitiator (polyethylene glycol, PEG) and subsequently grafted on to PVDF/SMA by alkyne-anhydride reaction...
August 31, 2017: Nanoscale
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...
August 10, 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
Sonia Del Prete, Rosa Perfetto, Mosè Rossi, Fatmah A S Alasmary, Sameh M Osman, Zeid AlOthman, Claudiu T Supuran, Clemente Capasso
The carbonic anhydrase superfamily (CA, EC of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9...
December 2017: Journal of Enzyme Inhibition and Medicinal Chemistry
Aatif Amin, Zakia Latif, Stanley J Opella
Mercury resistant (Hg(R)) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 μg/mL against mercuric chloride (HgCl2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury resistant Gram positive bacteria...
August 8, 2017: Journal of Microbiology and Biotechnology
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