STUDIES OF THE ANAEROBICALLY INDUCED PROMOTER PNIRB AND THE IMPROVED EXPRESSION OF BACTERIAL-ANTIGENS

The promoter of the Escherichia coli gene nirB is induced by both the presence of nitrite in the environment and by low oxygen tensions. It has been used to direct the high-level expression of heterologous prot eins by E. coli strains in fermenters, and attenuated Salmonella strai ns expressing foreign proteins under nirB promoter (pnir) control have efficiently induced an immune response against these proteins. The ge nes encoding two different E. coli envelope proteins, the outer membra ne protein LamB and the periplasmic protein MalE, were placed under pn ir control on pBR322 derivatives, and both proteins were expressed at high levels duping anaerobic growth. Our results showed that the expre ssion level of MalE was influenced by the distance between the pnir pr omoter and the Shine-Dalgarno sequence: the highest levels were obtain ed by the longest constructs made; pnir directed a 4-fold increase in the level of MalE expression relative to the level reached by the prev iously described ptac-MalE expression vector. The best pnir construct produced 25 mg of MalE protein per 5 x 10(11) bacteria, which represen ts over 20% of total cell protein. Overexpression of MalE was well tol erated by E. coli, even under strict anaerobic conditions; for lamB, o ptimal induction was achieved under partial anaerobiosis. A MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160 inserted in to site 133 of MalE) was also overexpressed at a similar yield under p nir control, without apparent degradation of the hybrid protein. Moreo ver, when expressed in attenuated aroA S. typhimurium strain SL3261, t he plasmids carrying malE and malE-HIV genes were stable in vitro and in vivo.