Applied Microbiology and Biotechnology

, Volume 45, Issue 1–2, pp 120–126

Temperature-regulated expression of the tac/lacI system for overproduction of a fungal xylanase in Escherichia coli

  • G. -P. Xue
  • J. S. Johnson
  • D. J. Smyth
  • L. M. Dierens
  • X. Wang
  • G. D. Simpson
  • K. S. Gobius
  • J. H. Aylward
Original Paper Applied Genetics and Regulations

Abstract

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl β-d-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λPl system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λPl-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperaturemodulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Boer HA de, Comstock, LJ and Vasser, M. (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80: 21–25CrossRefGoogle Scholar
  2. Calos MP (1978) DNA sequence for a low-level promoter of the lac repressor gene and an ‘up’ promoter mutation. Nature 274: 762–769CrossRefGoogle Scholar
  3. Cheng X and Patterson TA (1992) Construction and use of λPl promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences. Nucleic Acids Res 20: 4591 4598CrossRefGoogle Scholar
  4. Gralla JD (1991) Promoter recognition and mRNA initiation by Eschcherichia coli Eσ 70. In: Goeddel DV, Emr SD, Henner DJ, Gold L and Levinson AD (eds) Gene expression technology. Academic Press, San Diego, Calif, pp 37–54Google Scholar
  5. Rosenberg M, Ho Y-S, Shatzman A (1983) The use of pKC30 and its derivative for controlled expression of genes. Methods Enzymol 101: 123–155CrossRefGoogle Scholar
  6. Sakamoto S, Iljima M, Matsuzawa H, Ohta T (1994) Production of thermophilic protease by glucose-controlled fed-batch culture of recombinant of Escherichia coli. J Ferment Bioeng 78: 304–309CrossRefGoogle Scholar
  7. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  8. Smit MH de, Duin J van (1990) Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci USA 87: 7668–7672CrossRefGoogle Scholar
  9. Xue GP, Gobius KS, Orpin CG (1992) A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities. J Gen Microbiol 138: 2397–2403Google Scholar
  10. Xue GP, Denman SE, Glassop D, Johnson JS, Dierens LM, Gobius KS, Aylward JH (1995) Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli. J Biotechnol 38: 269–277CrossRefGoogle Scholar
  11. Yabuta M, Onai-Miura S, Ohsuye K (1995) Thermo-inducible expression of a recombinant fusion protein by Escherichia coli lac repressor mutants. J Biotechnol 39: 67–73CrossRefGoogle Scholar
  12. Yee L, Blanch HW (1992) Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology 10: 1550–1557CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • G. -P. Xue
    • 1
  • J. S. Johnson
    • 1
  • D. J. Smyth
    • 1
  • L. M. Dierens
    • 1
  • X. Wang
    • 1
  • G. D. Simpson
    • 1
  • K. S. Gobius
    • 1
  • J. H. Aylward
    • 1
  1. 1.CSIRO Division of Tropical Crops and PasturesSt. LuciaAustralia

Personalised recommendations