Cloning of group A streptococcal pyrogenic exotoxin-B gene and its recombinant protein expression in culture supernatant]

Streptococcal pyrogenic exotoxin B, a conserved cysteine protease (SPE B/SCP) released by group A Streptococcus (GAS) strains, is considered to be an important virulence factor of this bacterium. This paper reports the cloning of gene encoding SPE B/SCP. For production of recombinant SPE B/SCP (rSPE B/SCP), the primers specific for the SPE B/SCP gene (spe b) were designed based on its nucleotide sequence. Polymerase chain reaction (PCR) was performed with the genomic DNA of GAS strain NZ131 as a template. The amplified PCR products were purified and cloned into the pBluescript II SK(+) plasmid vector. The vector was transformed into Escherichia coli (E. coli) JM109. The rSPE B/SCP and its recombinant proenzyme (rzym) were secreted in the culture supernate of the transformant. The rSPE B/SCP was purified from the supernatant by sequential chromatography on DEAE-Sepharose, matrix gel Red A and Sephadex G-50 columns. The purified rzym and rSPE B/SCP, respectively, gave a single band with a molecular weight approximately 40 kDa and 27 kDa on SDS-polyacrylamide gel electrophoresis, and reacted with anti-SPE B/SCP antibodies in Western Blot analysis. This is the first report in which rSPE B/SCP was obtained from the culture supernate of the transformant.