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Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis

Paper ID Volume ID Publish Year Pages File Format Full-Text
24532 43524 2008 6 PDF Available
Title
Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis
Abstract

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, cspAΔCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspAΔCWA 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 CspAΔCWA. CspAΔCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspAΔCWA 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.

Keywords
GBS, group B Streptococcus (Streptococcus agalactiae); CspA, cell-surface protease A of Streptococcus agalactiae; THB, Todd–Hewitt Broth; CWA, cell-wall anchor; CEP, cell-envelope protease; MALDI-TOF, matrix-assisted laser desorption/ionization of time of
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Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 136, Issues 3–4, 10 September 2008, Pages 129–134
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us