Expression of Recombinant Proteins in Escherichia coli Using an Oxygen-Responsive Promoter

Article metrics


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 β-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that β-galactosidase was expressed to a level of approximately 10% of total cellular protein.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Yanisch-Perron, C., Vieira, J., Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119.

  2. 2

    Tacon, W., Carey, N. and Emtage, S. 1980. The construction and characterization of plasmid vectors suitable for the expression of all DNA phases under the control of the E. coli tryptophan promoter. Mol. Gen. Genet. 177: 427–438.

  3. 3

    de Boer, H.A., Comstock, L.J. and Vasser, M. 1983. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA. 80: 21–25.

  4. 4

    Remaut, E., Stanssens, P. and Fiers, W. 1981. Plasmid vectors for high-efficiency expression controlled by the p L promoter of coliphage lambda. Gene 15: 81–93.

  5. 5

    Shirakawa, M., Tsurimoto, T. and Matsubara, K. 1984. Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli. Gene 28: 127–132.

  6. 6

    Hoffman, C.S. and Wright, A. 1985. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc. Natl. Acad. Sci. USA. 82: 5107–5111.

  7. 7

    Tabor, S. and Richardson, C.C. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA. 82: 1074–1078.

  8. 8

    Khosla, C. and Bailey, J.E., 1988. Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli. Mol. Gen. Genet. 214: 158–161.

  9. 9

    Khosla, C. and Bailey, J.E. 1988. Heterologous expression of a bacterial hemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331: 633–635.

  10. 10

    Khosla, C. and Bailey, J.E. 1989. Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. J. Bacteriol. 171: 5995–6004.

  11. 11

    Anraku, Y. and Gennis, R.B. 1987. The aerobic respiratory chain of Escherichia coli. Trends Biochem. Sci. 12: 262–266.

  12. 12

    Zabriskie, D.W. and Arcuri, E.J. 1986. Factors influencing productivity of fermentations employing recombinant microorganisms. Enzyme Microb. Technol. 8: 706–717.

  13. 13

    Postma, P.W. 1987. Phosphotransferase system for glucose and other sugars, p. 127–141. In: Escherichia coli and Salmonella typhimurium. Neidhardt, F. C. (Ed.). American Society for Microbiology, Washington D.C.

  14. 14

    Schein, C.H. and Noteborn, M.H.M. 1988. Formation of recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6: 291–294.

  15. 15

    Seo, J.H. and Bailey, J.E. 1985. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol. Bioeng. 27: 1668–1674.

  16. 16

    Yamamoto, N. and Droffner, M.L. 1985. Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc. Natl. Acad. Sci. USA. 82: 2077–2081.

  17. 17

    Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

  18. 18

    Neumann, J.R., Morency, C.A. and Russian, K.O. 1987. A novel rapid assay for chloramphenicol acetyltransferase gene expression. Biotechniques 5: 444–447.

  19. 19

    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Further reading