Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis.
The heterologous expression of streptococcal genes in common Gram-negative hosts may be complicated by low-level expression, toxicity to the host, formation of inclusion bodies, and mislocalization of the encoded proteins. Biochemical study of the Streptococcus agalactiae virulence-associated cell-envelope protease (CEP) CspA, as well as other CEPs, has been limited by the lack of effective expression systems. In this study, we present a simple strategy to express cspA as a catalytically active exoprotein. A recombinant allele of cspA, cspADeltaCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspADeltaCWA allele was expressed in the Gram-positive organism, Lactococcus lactis, using an established, plasmid-based, nisin-inducible expression system. After induction, nearly all of the exoprotein observable by SDS-PAGE corresponded to CspADeltaCWA. CspADeltaCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspADeltaCWA indicated that like some other CEPs, it is N-terminally processed, exists predominantly as a dimer, and has the ability to cleave itself at its C-terminus. Taken together, this work presents an efficient strategy for expression of cspA that could be applied to other streptococcal proteins that are not amenable to expression using common Gram-negative hosts.
Shelver
Daniel
D
Louisiana State University Health Sciences Center in Shreveport, Department of Microbiology and Immunology, Shreveport, LA 71130, United States. dshelv@lsuhsc.edu
Bryan
Joshua D
JD
D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D012995:Solubility; D013292:Streptococcus agalactiae / Q000235:genetics*; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D037521:Virulence Factors; D012697:Serine Endopeptidases