Optimised Heterologous Expression and Functional Analysis ofthe Yersinia pestis F1-Capsular Antigen Regulator Caf1R

• Published in: International journal of molecular sciences 2021, Vol.22 (18)
• Main Authors: Kumar Gahlot, Dharmender, Ifill, Gyles, MacIntyre, Sheila
• Format: Article
• Language: English
• Subjects: Biochemistry; biokemi; Caf1R; F1 capsule; functional analysis; Microbiology; mikrobiologi; Molecular Biology; molekylärbiologi; transcriptional regulator; Yersinia pestis
• ISSN: 1661-6596; 1422-0067
• DOI: 10.3390/ijms22189805

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continuesto cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protectivecoat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to itsclinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightlyregulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone toaggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression fromthe native and synthetic codon-optimised caf1R cloned in three different expression plasmids wasexamined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstratedin vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R wereproduced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoterand Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identifiedCaf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correctfolding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availabilityof functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 andCaf1R-regulated pathophysiology of Y. pestis.