Optimised heterologous expression and functional analysis of the Yersinia pestis F1-Capsular antigen regulator Caf1R

Gahlot, D. ORCID: https://orcid.org/0000-0001-6963-0009, Ifill, G. and MacIntyre, S. (2021) Optimised heterologous expression and functional analysis of the Yersinia pestis F1-Capsular antigen regulator Caf1R. International Journal of Molecular Sciences, 22 (18). ISSN 1422-0067 doi: 10.3390/ijms22189805

Abstract/Summary

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/100228
Item Type	Article
Divisions	Life Sciences > School of Biological Sciences > Biomedical Sciences
Uncontrolled Keywords	Yersinia pestis, F1 capsule, transcriptional regulator, Caf1R, functional analysis
Publisher	MDPI
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