Optimised Heterologous Expression and Functional Analysis of the F1-Capsular Antigen Regulator Caf1R.

The bacterial pathogen, , has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, 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 cloned in three different expression plasmids was examined in a library of 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 . Transcriptional- reporter fusions defined the P promoter and Caf1R binding site responsible for transcription of the operon. Use of the identified Caf1R binding 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 .