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. 1991 May 11;19(9):2417–2421. doi: 10.1093/nar/19.9.2417

High level heterologous expression in E. coli using mutant forms of the lac promoter.

A J Makoff 1, M D Oxer 1
PMCID: PMC329451  PMID: 2041779

Abstract

Single base deletions in the lac promoter which reduced the 18bp spacing between the -35 and -10 homology regions to 17bp, increased the strength of the promoter. A single base substitution (T----G) in the -35 region to generate the consensus sequence TTG-ACA increased the strength further and no longer required a 17bp spacing. The mutated lac promoter was as powerful as a shorter form of the tac promoter which lacked two AT-rich regions upstream of the -35 region, and expressed the P69 surface antigen (pertactin) of Bordetella pertussis to 30-40% total cell protein and tetanus toxin fragment C to 16-20% total cell protein.

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Selected References

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  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Angus P. W., Mihaly G. W., Morgan D. J., Smallwood R. A. Oxygen dependence of omeprazole clearance and sulfone and sulfide metabolite formation in the isolated perfused rat liver. J Pharmacol Exp Ther. 1989 Sep;250(3):1043–1047. [PubMed] [Google Scholar]
  3. Aoyama T., Takanami M., Ohtsuka E., Taniyama Y., Marumoto R., Sato H., Ikehara M. Essential structure of E. coli promoter: effect of spacer length between the two consensus sequences on promoter function. Nucleic Acids Res. 1983 Sep 10;11(17):5855–5864. doi: 10.1093/nar/11.17.5855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brosius J., Erfle M., Storella J. Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity. J Biol Chem. 1985 Mar 25;260(6):3539–3541. [PubMed] [Google Scholar]
  5. Deuschle U., Kammerer W., Gentz R., Bujard H. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986 Nov;5(11):2987–2994. doi: 10.1002/j.1460-2075.1986.tb04596.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fuller F. A family of cloning vectors containing the lacUV5 promoter. Gene. 1982 Jul-Aug;19(1):43–54. doi: 10.1016/0378-1119(82)90187-1. [DOI] [PubMed] [Google Scholar]
  7. Kammerer W., Deuschle U., Gentz R., Bujard H. Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. EMBO J. 1986 Nov;5(11):2995–3000. doi: 10.1002/j.1460-2075.1986.tb04597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kobayashi M., Nagata K., Ishihama A. Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter -35 region on promoter strength. Nucleic Acids Res. 1990 Dec 25;18(24):7367–7372. doi: 10.1093/nar/18.24.7367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Makoff A. J., Oxer M. D., Ballantine S. P., Fairweather N. F., Charles I. G. Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli. Biotechnology (N Y) 1990 Nov;8(11):1030–1033. doi: 10.1038/nbt1190-1030. [DOI] [PubMed] [Google Scholar]
  10. Makoff A. J., Oxer M. D., Romanos M. A., Fairweather N. F., Ballantine S. Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic Acids Res. 1989 Dec 25;17(24):10191–10202. doi: 10.1093/nar/17.24.10191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Makoff A. J., Smallwood A. E. The use of two-cistron constructions in improving the expression of a heterologous gene in E. coli. Nucleic Acids Res. 1990 Apr 11;18(7):1711–1718. doi: 10.1093/nar/18.7.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Makoff A., Parry N., Dicken L. Translational fusions with fragments of the trpE gene improve the expression of a poorly expressed heterologous gene in Escherichia coli. J Gen Microbiol. 1989 Jan;135(1):11–24. doi: 10.1099/00221287-135-1-11. [DOI] [PubMed] [Google Scholar]
  13. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  14. Mulligan M. E., Brosius J., McClure W. R. Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter. J Biol Chem. 1985 Mar 25;260(6):3529–3538. [PubMed] [Google Scholar]
  15. Nishi T., Itoh S. Enhancement of transcriptional activity of the Escherichia coli trp promoter by upstream A + T-rich regions. Gene. 1986;44(1):29–36. doi: 10.1016/0378-1119(86)90039-9. [DOI] [PubMed] [Google Scholar]
  16. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  17. Russell D. R., Bennett G. N. Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing. Gene. 1982 Dec;20(2):231–243. doi: 10.1016/0378-1119(82)90042-7. [DOI] [PubMed] [Google Scholar]
  18. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  19. Stefano J. E., Gralla J. D. Spacer mutations in the lac ps promoter. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1069–1072. doi: 10.1073/pnas.79.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  21. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

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