EXPRESSION OF A-SUBUNIT AND B-SUBUNIT OF SHIGA-LIKE TOXIN-II AS FUSIONS WITH GLUTATHIONE-S-TRANSFERASE AND THEIR POTENTIAL FOR USE IN SEROEPIDEMIOLOGY

We used the plasmid vector pGEX-2T for the expression of recombinant s ubunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically f used to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. Th e subunit genes were constructed synthetically by polymerase chain rea ction, with appropriate restriction sites to permit in-frame downstrea m insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl- beta-D-thiogalactopyranoside (IPTG) yielded only small quantities of S LT-IIA fusion proteins. Since IPTG induction was lethal for cells harb oring pFG2, we constructed the recombinant plasmid pFG4, which contain ed a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatograph y on immobilized glutathione and used as an antigen in immunoblot anal ysis. Rabbit serum against native SLT-II, as well as all of 12 serum s amples with high neutralizing activity against SLT-II, reacted with SL T-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failu re of specific reactivity with the SLT-IIB fusion protein in the major ity of human serum samples with low neutralizing activity suggests tha t serum factors other than immunoglobulins may be responsible for neut ralizing activity in these cases. The immunoblot assay with recombinan t SLT-IIB as the antigen can be recommended for use in a diagnostic se tting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.