FIG. 1.

Construction of SpeB expression plasmids. The vectors are based on the speB gene of MGAS 1719 (15). This speB allele was previously subjected to site-specific mutagenesis to create a gene that produces a C192S mutant SpeB molecule (22). The entire open reading frame of the mutant gene was amplified by PCR from pSEBC2S (22) and cloned into the multiple-cloning site of the His-tag expression vector pPROEX-1 to yield pGM-1. pGM-5 is identical to pGM-1 except that it lacks an 81-bp segment encoding the 27-amino-acid (aa) secretion signal sequence. pGM-8, made by cloning speB codons 146 to 398 into pPROEX-1, expresses a 28-kDa mature form of SpeB containing the C192S amino acid substitution. pGM-6 was constructed by cloning speB codons 28 to 145 (encoding the propeptide fragment) into pPROEX-1. SP, signal peptide; PP, propeptide; MP, mature protein.