Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Dec 25;17(24):10191–10202. doi: 10.1093/nar/17.24.10191

Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.

A J Makoff 1, M D Oxer 1, M A Romanos 1, N F Fairweather 1, S Ballantine 1
PMCID: PMC335293  PMID: 2690015

Abstract

Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.

Full text

PDF
10198

Images in this article

10197Image
on p.10197
10200Image
on p.10200

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Bonekamp F., Andersen H. D., Christensen T., Jensen K. F. Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation. Nucleic Acids Res. 1985 Jun 11;13(11):4113–4123. doi: 10.1093/nar/13.11.4113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cannistraro V. J., Subbarao M. N., Kennell D. Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA. J Mol Biol. 1986 Nov 20;192(2):257–274. doi: 10.1016/0022-2836(86)90363-3. [DOI] [PubMed] [Google Scholar]
  5. Devos R., Plaetinck G., Cheroutre H., Simons G., Degrave W., Tavernier J., Remaut E., Fiers W. Molecular cloning of human interleukin 2 cDNA and its expression in E. coli. Nucleic Acids Res. 1983 Jul 11;11(13):4307–4323. doi: 10.1093/nar/11.13.4307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fairweather N. F., Lyness V. A. The complete nucleotide sequence of tetanus toxin. Nucleic Acids Res. 1986 Oct 10;14(19):7809–7812. doi: 10.1093/nar/14.19.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Gouy M., Gautier C. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 1982 Nov 25;10(22):7055–7074. doi: 10.1093/nar/10.22.7055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grosjean H., Fiers W. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982 Jun;18(3):199–209. doi: 10.1016/0378-1119(82)90157-3. [DOI] [PubMed] [Google Scholar]
  10. Helting T. B., Nau H. H. Analysis of the immune response to papain digestion products of tetanus toxin. Acta Pathol Microbiol Immunol Scand C. 1984 Feb;92(1):59–63. doi: 10.1111/j.1699-0463.1984.tb00052.x. [DOI] [PubMed] [Google Scholar]
  11. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J Mol Biol. 1981 Feb 15;146(1):1–21. doi: 10.1016/0022-2836(81)90363-6. [DOI] [PubMed] [Google Scholar]
  12. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol. 1981 Sep 25;151(3):389–409. doi: 10.1016/0022-2836(81)90003-6. [DOI] [PubMed] [Google Scholar]
  13. Ivanoff L. A., Towatari T., Ray J., Korant B. D., Petteway S. R., Jr Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5392–5396. doi: 10.1073/pnas.83.15.5392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Larder B., Purifoy D., Powell K., Darby G. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli. EMBO J. 1987 Oct;6(10):3133–3137. doi: 10.1002/j.1460-2075.1987.tb02623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Miki T., Yasukochi T., Nagatani H., Furuno M., Orita T., Yamada H., Imoto T., Horiuchi T. Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme. Protein Eng. 1987 Aug-Sep;1(4):327–332. doi: 10.1093/protein/1.4.327. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. O'Neill G. P., Kilburn D. G., Warren R. A., Miller R. C., Jr Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli. Appl Environ Microbiol. 1986 Oct;52(4):737–743. doi: 10.1128/aem.52.4.737-743.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pedersen S. Escherichia coli ribosomes translate in vivo with variable rate. EMBO J. 1984 Dec 1;3(12):2895–2898. doi: 10.1002/j.1460-2075.1984.tb02227.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pennica D., Hayflick J. S., Bringman T. S., Palladino M. A., Goeddel D. V. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6060–6064. doi: 10.1073/pnas.82.18.6060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  24. Robinson M., Lilley R., Little S., Emtage J. S., Yarranton G., Stephens P., Millican A., Eaton M., Humphreys G. Codon usage can affect efficiency of translation of genes in Escherichia coli. Nucleic Acids Res. 1984 Sep 11;12(17):6663–6671. doi: 10.1093/nar/12.17.6663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schoemaker J. M., Brasnett A. H., Marston F. A. Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. EMBO J. 1985 Mar;4(3):775–780. doi: 10.1002/j.1460-2075.1985.tb03696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seeburg P. H., Sias S., Adelman J., de Boer H. A., Hayflick J., Jhurani P., Goeddel D. V., Heyneker H. L. Efficient bacterial expression of bovine and porcine growth hormones. DNA. 1983;2(1):37–45. doi: 10.1089/dna.1.1983.2.37. [DOI] [PubMed] [Google Scholar]
  28. Sekine S., Mizukami T., Nishi T., Kuwana Y., Saito A., Sato M., Itoh S., Kawauchi H. Cloning and expression of cDNA for salmon growth hormone in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4306–4310. doi: 10.1073/pnas.82.13.4306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sharp P. M., Li W. H. Codon usage in regulatory genes in Escherichia coli does not reflect selection for 'rare' codons. Nucleic Acids Res. 1986 Oct 10;14(19):7737–7749. doi: 10.1093/nar/14.19.7737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Simons G., Remaut E., Allet B., Devos R., Fiers W. High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda. Gene. 1984 Apr;28(1):55–64. doi: 10.1016/0378-1119(84)90087-8. [DOI] [PubMed] [Google Scholar]
  31. Stanssens P., Remaut E., Fiers W. Inefficient translation initiation causes premature transcription termination in the lacZ gene. Cell. 1986 Mar 14;44(5):711–718. doi: 10.1016/0092-8674(86)90837-8. [DOI] [PubMed] [Google Scholar]
  32. Sørensen M. A., Kurland C. G., Pedersen S. Codon usage determines translation rate in Escherichia coli. J Mol Biol. 1989 May 20;207(2):365–377. doi: 10.1016/0022-2836(89)90260-x. [DOI] [PubMed] [Google Scholar]
  33. Tanizawa Y., Kishi F., Kaneko T., Nakazawa A. High level expression of chicken muscle adenylate kinase in Escherichia coli. J Biochem. 1987 May;101(5):1289–1296. doi: 10.1093/oxfordjournals.jbchem.a121993. [DOI] [PubMed] [Google Scholar]
  34. Tucker J., Sczakiel G., Feuerstein J., John J., Goody R. S., Wittinghofer A. Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 1986 Jun;5(6):1351–1358. doi: 10.1002/j.1460-2075.1986.tb04366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Varenne S., Buc J., Lloubes R., Lazdunski C. Translation is a non-uniform process. Effect of tRNA availability on the rate of elongation of nascent polypeptide chains. J Mol Biol. 1984 Dec 15;180(3):549–576. doi: 10.1016/0022-2836(84)90027-5. [DOI] [PubMed] [Google Scholar]
  36. Varenne S., Lazdunski C. Effect of distribution of unfavourable codons on the maximum rate of gene expression by an heterologous organism. J Theor Biol. 1986 May 7;120(1):99–110. doi: 10.1016/s0022-5193(86)80020-0. [DOI] [PubMed] [Google Scholar]
  37. Whitehorn E. A., Livak K. J., Petteway S. R., Jr The effects of hybrid ribosome-binding-site variants on the expression of human interferon-beta in Escherichia coli. Gene. 1985;36(3):375–379. doi: 10.1016/0378-1119(85)90194-5. [DOI] [PubMed] [Google Scholar]
  38. Williams D. P., Regier D., Akiyoshi D., Genbauffe F., Murphy J. R. Design, synthesis and expression of a human interleukin-2 gene incorporating the codon usage bias found in highly expressed Escherichia coli genes. Nucleic Acids Res. 1988 Nov 25;16(22):10453–10467. doi: 10.1093/nar/16.22.10453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wong E. M., Muesing M. A., Polisky B. Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3570–3574. doi: 10.1073/pnas.79.11.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young J. F., Desselberger U., Palese P., Ferguson B., Shatzman A. R., Rosenberg M. Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6105–6109. doi: 10.1073/pnas.80.19.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES