Two types of genetically mutated toxoids have been evaluated. One is the B subunit [12-14] and the other is attenuated holotoxin, which contains one or two mutations in the active center of the A subunit. The advantage of

the B subunit vaccine is its safety, which is attributable to a total lack of the A subunit. On the other hand, genetically mutated holotoxoids are beneficial because they safely induce anti-A subunit antibody Kinase Inhibitor Library production. The enzymatic activity of the A subunit is reportedly reduced by mutations at position 167 (glutamic acid to glutamine), 170 (arginine to leucine), or both [15-18]. Additionally, a number of reports have shown that genetically attenuated holotoxins, such as mutant Stx1 [19, 20], mStx2 [20], mutant hybrid proteins [21], and mStx2e [22-24], are good candidates for vaccine antigens for prevention of Shiga toxemia. However, because the purification yields described in some reports are far too small for the practical use of these toxoids, overexpression and purification methods need to

be developed for these antigen proteins. We previously reported an overexpression method for production of recombinant CTB in E. coli [25]. In the expression plasmid, the entire CTB gene was inserted into the lacZα gene of a pBluescript II SK(+) vector with a Shine-Dalgarno sequence derived selleck products from the LTB of enterotoxigenic E. coli. Protein expression was induced only by cultivating the K12 derivative E. coli strain MV1184 transformed with the expression plasmid in CAYE broth containing lincomycin, which was originally identified Tryptophan synthase as an antibiotic that prevents protein synthesis

in gram-positive bacteria through inhibition of peptidyltransferase activity on the 50S ribosomal subunit [26]. Because this expression method has also been successfully applied to overexpression of CT [25], we reasoned that it would be applicable to overexpression of Stx, especially wild-type and mStx2. In this paper, we present a lincomycin-inducible overexpression method for production of Stx2 and its mutant proteins. These proteins were expressed as histidine-tag fusion proteins at the C-terminal ends of the B subunits (Stx2-His and mStx2-His, respectively). We demonstrate the safety and antigenicity of mStx2-His as a vaccine antigen to protect mice from Shiga toxemia. The expression plasmid for Stx2-His was prepared according to a previously published procedure for CT preparation [25]. The complete nucleotide sequence of the gene encoding Stx2 was PCR amplified using the genomic DNA of E. coli O157:H7 (which was an outbreak strain in Okayama, Japan in 1996) as template DNA and a set of two primers, LTB(SD)Stx2(EcoRI)-f and Stx2B(6 x His)HindIII-r. The forward primer included the SD sequence derived from LTB upstream from the start codon of the Stx2 gene and the reverse primer was a fusion of the end of the B subunit gene and six-histidine (6 x His)-coding sequences. The amplified product was cloned into the pCR2.