Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter.
The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.
Khosla
C
C
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125.
Curtis
J E
JE
Bydalek
P
P
Swartz
J R
JR
Bailey
J E
JE
D001419:Bacteria / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005990:Glycerol / Q000494:pharmacology; D006454:Hemoglobins / Q000096:biosynthesis / Q000235:genetics*; D009584:Nitrogen / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D006454:Hemoglobins; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D009584:Nitrogen; D005990:Glycerol; D010100:Oxygen