Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions

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: 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.

Molecular mechanisms of vancomycin resistance

“…VanZ A does not affect the population of UDP‐MurNAc‐pentapeptide[ d ‐Ala] or UDP‐MurNAc‐pentapeptide[ d ‐lac] and its role in the Van cascade is currently not known . VanZ A was recently purified to homogeneity and its localization to the membrane validated …”

“…21,22 VanZ A was recently purified to homogeneity and its localization to the membrane validated. 69 Of note, a second vanZ was discovered in a VanF-type (D-Ala-D-Lac) resistance cassette in Paenibacillus papillae. 70 The corresponding VanZ F protein shared 21% primary sequence identity with VanZ A but the significance of this protein in the resistance cascade remains unknown.…”

The gelatinase biosynthesis‐activating pheromone binds and stabilises the FsrB membrane protein in Enterococcus faecalis quorum sensing

“…Purification of FsrB via the hexa‐Histidine tag engineered at the C terminus of the protein was achieved using well‐established methods developed in our laboratory for members of a range of membrane protein classes ). Figure summarises the successful application of these methods for production of FsrB.…”

“…The plasmid pTTQ18His‐FsrB was transformed into Escherichia coli BL21 [DE3]. For preparation of purified FsrB, 6 L volumes of E. coli BL21 [DE3]/pTTQ18His‐FsrB were cultured at 37 °C in Luria–Bertani broth containing 100 μg·mL −1 carbenicillin as described previously . Expression of fsrB was induced using 0.2 m m isopropyl thiogalactoside (IPTG) and cultures were incubated at an optimised postinduction temperature of 33 °C for a further 3 h prior to harvesting.…”

Membrane Sensor Histidine Kinases: Insights from Structural, Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

“…The advantage of this plasmid is that expression should be possible in almost any suitably engineered E. coli host independently of T7 polymerase-dependent systems. Use of pTTQ18His as expression plasmid has resulted in a high degree of success for producing intact SHKs compared with members of other membrane protein families, including successful expression of 15 of the 16 genome complement of membrane SHKs of Enterococcus faecalis [ 34 ], VanS (A-type) of E. faecium [ 75 , 89 ], BlpH, and ComD2 pheromone-sensing SHKs of Streptococcus pneumoniae (together with several other membrane proteins associated with antimicrobial resistance) [ 90 ] and the PrrB (or RegB) redox-sensing SHK protein of the Gram-negative bacterium Rhodobacter sphaeroides [ 87 , 91 ]. Other membrane overexpression plasmids include pT7-7 [ 92 ] based on T7 polymerase [ 93 ], pProEX available commercially [ 94 ] and pETCH [ 95 ] derived from SUMO fusion vectors [ 96 ].…”