Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Gene
G D WestropJ Coote

Abstract

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vit...Continue Reading

References

May 5, 1991·Journal of Molecular Biology·F W Studier
Mar 1, 1991·Molecular Microbiology·R A Welch
Apr 27, 1989·Nature·W R Brown
May 5, 1986·Journal of Molecular Biology·F W Studier, B A Moffatt
Aug 1, 1984·The Journal of Infectious Diseases·A A WeissS Falkow
Jan 1, 1981·Methods in Enzymology·C O RockJ E Cronan

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Citations

Jul 27, 2007·Natural Product Reports·Andrew C Mercer, Michael D Burkart
Feb 7, 2004·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·James D CherryMichael J Corbel
Sep 2, 2015·Biochemical and Biophysical Research Communications·Riyaz Ahmad PanditChanan Angsuthanasombat
Sep 26, 2013·The Journal of Biological Chemistry·Orso SubriniAlexandre Chenal
Jun 22, 2019·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Darragh P O'BrienAlexandre Chenal
Jan 18, 2018·Toxicon : Official Journal of the International Society on Toxinology·Darragh P O'BrienAlexandre Chenal

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