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Article

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

1
School of Biological Sciences, University of Reading, Reading RG6 6UR, UK
2
Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden
3
Umeå Centre for Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden
4
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
*
Authors to whom correspondence should be addressed.
Academic Editors: Neus Ferrer-Miralles and Joaquin Seras-Franzoso
Int. J. Mol. Sci. 2021, 22(18), 9805; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189805
Received: 2 August 2021 / Revised: 2 September 2021 / Accepted: 6 September 2021 / Published: 10 September 2021
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. View Full-Text
Keywords: Yersinia pestis; F1 capsule; transcriptional regulator; Caf1R; functional analysis Yersinia pestis; F1 capsule; transcriptional regulator; Caf1R; functional analysis
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MDPI and ACS Style

Gahlot, D.K.; Ifill, G.; MacIntyre, S. Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R. Int. J. Mol. Sci. 2021, 22, 9805. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189805

AMA Style

Gahlot DK, Ifill G, MacIntyre S. Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R. International Journal of Molecular Sciences. 2021; 22(18):9805. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189805

Chicago/Turabian Style

Gahlot, Dharmender K., Gyles Ifill, and Sheila MacIntyre. 2021. "Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R" International Journal of Molecular Sciences 22, no. 18: 9805. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189805

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