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 proteins by E. coli strains in fermentors, and attenuated Salmonella strains expressing foreign proteins under nirB promoter (pnir) control have efficiently induced an immune response against these proteins. The genes encoding two different E. coli envelope proteins, the outer membrane protein LamB and the periplasmic protein MalE, were placed under pnir control on pBR322 derivatives, and both proteins were expressed at high levels during anaerobic growth. Our results showed that the expression level of MalE was influenced by the distance between the pnir promoter and the Shine-Dalgarno sequence: the highest levels were obtained by the longest constructs made; pnir directed a 4-fold increase in the level of MalE expression relative to the level reached by the previously described ptac-MalE expression vector. The best pnir construct produced 25 mg of MalE protein per 5 x 10(11) bacteria, which represents over 20% of total cell protein. Overexpression of MalE was well tolerated by E. coli, even under strict anaerobic conditions; for LamB, optimal induction was achieved under partial anaerobiosis. A MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160 inserted into site 133 of MalE) was also overexpressed at a similar yield under pnir control, without apparent degradation of the hybrid protein. Moreover, when expressed in attenuated aroA S. typhimurium strain SL3261, the plasmids carrying malE and malE-HIV genes were stable in vitro and in vivo.