Expression of A and B subunits 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 subunits 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 fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream 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) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained 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 chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-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. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.
Gunzer
F
F
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany.
Karch
H
H
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000472:pathogenicity*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006463:Hemolytic-Uremic Syndrome / Q000175:diagnosis; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D012698:Serologic Tests; D022641:Shiga Toxin 2
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; D022641:Shiga Toxin 2; D005982:Glutathione Transferase