CONSTRUCTION AND CHARACTERIZATION OF VERSATILE CLONING VECTORS FOR EFFICIENT DELIVERY OF NATIVE FOREIGN PROTEINS TO THE PERIPLASM OF ESCHERICHIA-COLI

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yield s of cholera toxin (CT). We found that production of wild type CT or i ts B subunit (CT-B) as a periplasmic protein was toxic for E. coli, bu t by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enteroto xin LTIIb, we succeeded for the first time in producing CT holotoxin i n high yield in E. coli. Based on these findings, we designed and cons tructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the per iplasm of E. coli. We confirmed the usefulness of these vectors by pro ducing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fo ld greater when the LTIIb-B signal sequence was used than when the nat ive leader for alkaline phosphatase was used. Second, using the pspA g ene that encodes pneumococcal surface protein A from Streptococcus pne umoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed t hat it was immunoreactive in Western blots with antibodies against nat ive PspA. The vectors described here will be useful for further studie s on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli. (C) 1997 Academic Press.