We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.Streptococcal pyrogenic exotoxin B (SPE B), known as streptococcal cysteine protease (SPE B/SCP) is highly conserved among group A Streptococcus (GAS). The structural gene, spe b, is found among all GAS clinical isolates (20) and expressed in almost all strains (12). This protein is initially secreted as a 42-kDa precursor, zymogen, and is autocatalytically cleaved to a 28-kDa mature SPE B/SCP (7, 9). SPE B/SCP cleaves or degrades fibronectin and vitronectin (12), which function as human matrix proteins. It also cleaves plasma kininogen and releases kinin (10) and cleaves the interleukin-1 (IL-1) precursor to the active form of IL-1 (12). Furthermore, SPE B/SCP activates the human matrix metalloprotease that increases type IV collagen degradation (2). Finally, genetically inactivated SPE B/SCP is attenuated in the mouse lethality model (15, 16). These findings suggest that SPE B/SCP activates inflammatory responses in the host and plays a very important role in pathogenesis of infections.The SPE B/SCP gene was previously cloned, sequenced (1, 9, 18), and expressed in Escherichia coli (6,8,17,19). However, the secretion of the active form of the protease from E. coli and its activity relative to the native molecule have not been examined. In order to clarify this question, we constructed an expression vector including spe b and its predicted promoter regions for transformation into E. coli. As a result, recombinant SPE B/SCP (rSPE B/SCP), retaining cysteine protease activity, was obtained from the culture supernatant.GAS strain NZ131 (kindly provided by D. R. Martin, NewZealand Communicable Disease Center, Porirua) chromosomal DNA was used as the template. Two oligonucleotides (SPEBF0008, 5ЈGTGTCAACTAACCGTGTTATTG-3Ј; SPEBR1485, 5Ј-TGATCTGTGTCTGASTGGATACTT-3Ј) were designed based on the spe b sequence as reported by Hauser et al. (9) and used as primers. PCR was performed with 25 cycles (94°C for 30 s, 54°C for 30 s, and 75°C for 1.5 min) and pyrobest DNA polymerase (TaKaRa Biomedicals, Kyoto, Japan). In order to construct the expression vector pSK-SCP, a 1,469-bp fragment including spe b and its predicted promoter region was amplified and cloned via the SmaI site in pBluescript II SK(ϩ) plasmid vector. pSK-SCP was transformed into E. coli strain JM109. The rSPE B/SCP was induced with 2 mM isopropyl--D-thiogalactopyranoside (IPTG; Wako Pure Chemical Co...