A NEW MEMBRANE-BOUND OPRI LIPOPROTEIN EXPRESSION VECTOR HIGH PRODUCTION OF HETEROLOGOUS FUSION PROTEINS IN GRAM (-) BACTERIA AND THE IMPLICATIONS FOR ORAL VACCINATION

We have previously described the development of cloning vectors for th e production of OprI-based outer membrane fusion proteins in E. coli ( Cornelis et al., 1996) and now describe the construction of a new vect or, containing a lacl(q) gene, resulting in tight repression of the pr omotor and allowing its use in other Gram (-) bacteria. The new pVUB3 expression vector encodes a truncated but active LacI(q'(341)) repress or which binds to the single operator in the vector. A high repression of the trc promotor was observed, resulting in a very low basal leaka ge of expression and very high production levels of OprI or derivative s after IPTG induction in E. coli. Bacterial viability was not affecte d under uninduced conditions, but the number of viable cell counts dec reased after production of large amounts of the outer membrane-bound O prI lipoprotein and its derivatives, both in E. coli and Salmonella Ty phimurium. This highly repressible system allows us to extend the use of OprI vectors in other Gram (-) bacteria, resulting in the productio n of outer membrane-bound lipid-modified molecules, opening the possib ility for its application in the design of potential live Salmonella-b ased subunit vaccines. (C) 1998 Elsevier Science B.V. All rights reser ved.