Azam, 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. and Phillips-Jones, Mary K. (2018) Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions. European Biophysics Journal, 47 (7). pp. 723-737. ISSN 0175-7571 (Print), 1432-1017 (Online) (doi:https://doi.org/10.1007/s00249-018-1325-z)

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Abstract

AbstractDespite 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: VanTG, VanTG-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 VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. 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.

Item Type:	Article
Additional Information:	© The Author(s) 2018. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Uncontrolled Keywords:	Histidine kinase, Membrane proteins, Vancomycin resistance, Enterococci, Pheromone sensors, Streptococcus pneumoniae, Circular dichroism spectroscopy, Analytical ultracentrifugation
Subjects:	Q Science > Q Science (General)
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
SWORD Depositor:	Users 6393 not found.
Last Modified:	22 Jun 2020 03:15
URI:	http://gala.gre.ac.uk/id/eprint/21764

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