Advertisement

Effects of temperature shift strategies on human preproinsulin production in the fed-batch fermentation of recombinantEscherichia coli

  • Young-Jin Son
  • Kyong-Hee Park
  • Sang-Yong Lee
  • Sung-Jin Oh
  • Chang-Kyu Kim
  • Byoung-Taek Choi
  • Yong-Cheol Park
  • Jin-Ho Seo
Article

Abstract

Preproinsulin is a well-known precursor of human insulin for the regulation of blood glucose levels. In this study, fed-batch fermentations of recombinantEscherichia coli JM109/pPT-MRpi were carried out for the overexpression of human preproinsulin. The expression of human preproinsulin was controlled by the temperature inducibleP2 promoter. The time-course profiles of fed-batch fermentation and SDS-PAGE analysis showed that human insulin expression was triggered by a culture temperature change from 30 to 37°C. Fermentation shift strategies, including the multi-step increase of temperature and the modulation of initiation time, were optimized to obtain high titers of cell mass and preproinsulin. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from 30 to 37°C for 2 h, gave the best results of 43.1 g/L of dry cell weight and 33.3% preproinsulin content, which corresponded to 2.0- and 1.2-fold increases, respectively, as compared to those of fed-batch culture at a constant temperature of 37°C.

Keywords

temperature shift fermentation human preproinsulin Escherichia coli fed-batch fermentation 

References

  1. 1.
    Barfoed, H. C. (1987) Insulin production technology.Chem. Eng. Prog. 83: 49–54.Google Scholar
  2. 2.
    Norman, A. and G. Litwack (1987)Hormones. 1st ed., pp. 122–123. Academic Press, New York, NY, USA.Google Scholar
  3. 3.
    Ladisch, M. R. and K. L. Kohlmann (1992) Recombinant human insulin.Biotechnol. Prog. 8: 469–478.CrossRefGoogle Scholar
  4. 4.
    Castellanos-Serra, L. R., E. Hardy, R. Ubieta, N. S. Vispo, C. Fernandez, V. Besada, V. Falcon, M. Gonzalez, A. Santos, G. Perez, A. Silva, and L. Herrera (1996) Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.FEBS Lett. 378: 171–176.CrossRefGoogle Scholar
  5. 5.
    Evans, D. B., W. G. Tarpley, and S. K. Sharma (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage.Protein Expr. Purif. 2: 205–213.CrossRefGoogle Scholar
  6. 6.
    Na, K. I., M. D. Kim, W. K. Min, J. A. Kim, W. J. Lee, D. O. Kim, K. M. Park, and J. H. Seo (2005) Expression and purification of ubiquitin-specific protease (UBP1) ofSaccharomyces cerevisiae in recombinantEscherichia coli.Biotechnol. Bioprocess Eng. 10:599–602.CrossRefGoogle Scholar
  7. 7.
    Sharma, S. K., D. B. Evans, A. F. Vosters, T. J. McQuade, and W. G. Tarpley (1991) Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain.Biotechnol. Appl. Biochem. 14:69–81.Google Scholar
  8. 8.
    Park, Y. C., S. J. Kim, J. H. Choi, W. H. Lee, K. M. Park, M. Kawamukai, Y. W. Ryu, and J. H. Seo (2005) Batch and fed-batch production of coenzyme Q(10) in recombinantEscherichia coli containing the decaprenyl diphosphate synthase gene fromGluconobacter suboxydans.Appl. Microbiol. Biotechnol. 67: 192–196.CrossRefGoogle Scholar
  9. 9.
    Wang, Y., P. Du, R. Gan, Z. Li, and Q. Ye (2005) Fedbatch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor.Biotechnol. Bioprocess Eng. 10: 149–154.CrossRefGoogle Scholar
  10. 10.
    Matsui, T., H. Yokota, S. Sato, S. Mukataka, and J. Takahashi (1989) Pressurized culture ofEscherichia coli for a high concentration.Agric. Biol. Chem. 53: 2115–2120.Google Scholar
  11. 11.
    Strandberg, L. and S. O. Enfors (1991) Batch and fed batch cultivations for the temperature induced production of a recombinant protein inEscherichia coli.Biotechnol. Lett. 13: 609–614.CrossRefGoogle Scholar
  12. 12.
    Jensen, E. B. and S. Carlsen (1990) Production of recombinant human growth hormone inEscherichia coli: Expression of different precursors and physiological effects of glucose, acetate, and salts.Biotechnol. Bioeng. 36: 1–11.CrossRefGoogle Scholar
  13. 13.
    Lan, J. C., T. C. Ling, G. Hamilton, and A. Lyddiatt (2006) A fermentation strategy for anti-MUC1 C595 diabody expression in recombinantEscherichia coli.Biotechnol. Bioprocess Eng. 11: 425–431.CrossRefGoogle Scholar
  14. 14.
    Luli, G. W. and W. R. Strohl (1990) Comparison of growth, acetate production, and acetate inhibition ofEscherichia coli strains in batch and fed-batch fermentations.Appl. Environ. Microbiol. 56: 1004–1011.Google Scholar
  15. 15.
    Gleiser, I. E. and S. Bauer (1981) Growth ofEscherichia coli W to high cell concentration by oxygen level linked control of carbon source concentration.Biotechnol. Bioeng. 23: 1015–1021.CrossRefGoogle Scholar
  16. 16.
    Rinas, U., H. A. Kracke-Helm, and K. Schugerl (1989) Glucose as a substrate in recombinant strain fermentation technology: by-product formation, degradation and intracellular accumulation of recombinant protein.Appl. Microbiol. Biotechnol. 31: 163–167.CrossRefGoogle Scholar
  17. 17.
    Yee, L. and H. W. Blanch (1993) Recombinant trypsin production in high cell density fed-batch cultures inEscherichia coli.Biotechnol. Bioeng. 41: 781–790.CrossRefGoogle Scholar
  18. 18.
    Birch, R. M. and G. M. Walker (2000) Influence of magnesium ions on heat shock and ethanol stress responses ofSaccharomyces cerevisiae.Enzyme Microb. Technol. 26: 678–687.CrossRefGoogle Scholar
  19. 19.
    Wouters, J. A., H. H. Kamphuis, J. Hugenholtz, O. P. Kuipers, W. M. de Vos, and T. Abee (2000) Changes in glycolytic activity ofLactococcus lactis induced by low temperature.Appl. Environ. Microbiol. 66: 3686–3691.CrossRefGoogle Scholar
  20. 20.
    Buttrick, P. (2006) The regulation of heat shock protein expression: How, when and where.J. Mol. Cell. Cardiol. 41: 785–786.CrossRefGoogle Scholar
  21. 21.
    Lamotte, D., M. Ouzzine, S. Fournel-Gigleux, J. Magdalou, and J. Boudrant (1996) A temperature profile in batch culture to increase the production of the recombinant UDP-glucuronosyltransferase 2B4 inEscherichia coli.Process Biochem. 31: 235–241.CrossRefGoogle Scholar
  22. 22.
    Lukacsovich, T., G. Baliko, A. Orosz, E. Balla, and P. Venetianer (1990) New approaches to increase the expression and stability of cloned foreign genes inEscherichia coli.J. Biotechnol. 13: 243–250.CrossRefGoogle Scholar
  23. 23.
    Rasmussen, L. J., A. Lobner-Olesen, and M. G. Marinus (1995) Growth-rate-dependent transcription initiation from the dam P2 promoter.Gene 157: 213–215.CrossRefGoogle Scholar
  24. 24.
    Murray, H. D., J. Alex Appleman, and R. L. Gourse (2003) Regulation of theEscherichia coli rrnB P2 promoter.J. Bacteriol. 185: 28–34.CrossRefGoogle Scholar
  25. 25.
    Kim, S. G., D. H. Kweon, D. H. Lee, Y. C. Park, and J. H. Seo (2005) Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase ofBacillus macerans in recombinantEscherichia coli.Protein Expr. Purif. 41: 426–432.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 2007

Authors and Affiliations

  • Young-Jin Son
    • 1
    • 2
  • Kyong-Hee Park
    • 3
  • Sang-Yong Lee
    • 2
  • Sung-Jin Oh
    • 2
  • Chang-Kyu Kim
    • 2
  • Byoung-Taek Choi
    • 2
  • Yong-Cheol Park
    • 4
  • Jin-Ho Seo
    • 1
  1. 1.Interdisciplinary Program of BioengineeringSeoul National UniversitySeoulKorea
  2. 2.Biotechnology Laboratory, CKDBiO Research InstituteCKDBiOAnsanKorea
  3. 3.Health Industry CenterChungbuk TechnoParkChungbukKorea
  4. 4.Center for Agricultural BiomaterialsSeoul National UniversitySeoulKorea

Personalised recommendations