A novel Escherichia coli vector for oxygen-inducible high level expression of foreign genes.
By utilizing the oxygen-sensitive Escherichia coli Mn-superoxide dismutase (Mn-SOD) promoter, we have developed a vector system that expresses high levels of cloned foreign genes. The promoter for the bacterial Mn-SOD, as well as both 5'-untranslated and transcriptional termination sequences were ligated to a synthetic linker containing two restriction 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-generating agent paraquat (methyl viologen) as the inducer. To test the vector, both native and mutated human Mn-SOD cDNAs were cloned and expressed, respectively. To determine the optimal concentration of inducer necessary for maximal expression, recombinant bacteria were exposed to increasing concentrations of paraquat and subsequently assayed for superoxide dismutase (SOD) activity. The highest expression was induced by 20 microM paraquat, and approached 50% of total soluble protein.
Gao
B
B
Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, Denver 80262, USA.
Flores
S C
SC
Bose
S K
SK
McCord
J M
JM
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008345:Manganese; D010100:Oxygen; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008345:Manganese; D013482:Superoxide Dismutase; D010100:Oxygen