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Eur Biophys J

Title:		Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions
Author(s):		Azam AA; Kinder JM; Khan GN; Alase A; Ma P; Liu Y; Ault JR; Henderson PJF; Chowdhry BZ; Alexander BD; Harding SE; Phillips-Jones MK;
Address:		"AMR Biophysics Group, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK. Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. Department of Pharmaceutical, Chemical and Environmental Sciences, University of Greenwich, Central Avenue, Chatham, Kent, ME4 4TB, UK. National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, UK. AMR Biophysics Group, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK. sbamp5@exmail.nottingham.ac.uk. National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, UK. sbamp5@exmail.nottingham.ac.uk"
Journal Title:		Eur Biophys J
Year:		2018
Volume:		20180731
Issue:		7
Page Number:		723 - 737
DOI:		10.1007/s00249-018-1325-z
ISSN/ISBN:		1432-1017 (Electronic) 0175-7571 (Print) 0175-7571 (Linking)
Abstract:		"Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanT(G), VanT(G)-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicK(ESD) of Enterococcus faecalis VicK. Purified VicK(ESD) possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicK(ESD) denaturation occurs between 15 and 50 degrees C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicK(ESD). Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies"
Keywords:		"Amino Acid Sequence Anti-Bacterial Agents/*pharmacology *Drug Resistance, Bacterial Membrane Proteins/chemistry/*metabolism Pheromones/*metabolism Protein Denaturation Solubility Temperature Analytical ultracentrifugation Circular dichroism spectroscopy E;"
Notes:		"MedlineAzam, Aalishaa A Kinder, Jean M Khan, G Nasir Alase, Ade Ma, Pikyee Liu, Yang Ault, James R Henderson, Peter J F Chowdhry, Babur Z Alexander, Bruce D Harding, Stephen E Phillips-Jones, Mary K eng BB/D001641/1/Biotechnology and Biological Sciences Research Council/United Kingdom Wellcome Trust/United Kingdom BB/M013081/1/Biotechnology and Biological Sciences Research Council/United Kingdom 094232/Wellcome Trust/United Kingdom Innovation Fund/University of Central Lancashire/ Germany 2018/08/02 Eur Biophys J. 2018 Oct; 47(7):723-737. doi: 10.1007/s00249-018-1325-z. Epub 2018 Jul 31"