A NOVEL ESCHERICHIA-COLI VECTOR FOR OXYGEN-INDUCIBLE HIGH-LEVEL EXPRESSION OF FOREIGN GENES

By utilizing the oxygen-sensitive Escherichia coli Mn-superoxide dismu tase (Mn-SOD) promoter, we have developed a vector system that express es high levels of cloned foreign genes. The promoter for the bacterial Mn-SOD, as well as both 5'-untranslated and transcriptional terminati on sequences were ligated to a synthetic linker containing two restric tion enzyme cloning sites. The vector also contained the gene for beta -lactamase, which confers ampicillin resistance to the host bacterium and provides a selectable marker. After screening and selection, high level of expression was achieved by exposure to the superoxide-generat ing agent paraquat (methyl viologen) as the inducer. To test the vecto r, both native and mutated human Mn-SOD cDNAs were cloned and expresse d, respectively. To determine the optimal concentration of inducer nec essary for maximal expression, recombinant bacteria were exposed to in creasing concentrations of paraquat and subsequently assayed for super oxide dismutase (SOD) activity. The highest expression was induced by 20 mu M paraquat, and approached 50% of total soluble protein.