Applied Microbiology and Biotechnology

, Volume 39, Issue 4–5, pp 541–546 | Cite as

Recombinant protein production in cultures of an Escherichia coli trp strain

  • G. Gosset
  • R. de Anda
  • N. Cruz
  • A. Martínez
  • R. Quintero
  • F. Bolivar
Applied Genetics and Regulation

Abstract

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Balbás P, Anda R de, Flores N, Alvarado X, Cruz N, Valle F, Bolivar F (1988) Overproduction of proteins by recombinant DNA: human insulin. In: Cañedo L, Todd L, Jaz J, Parker L (eds) Cell function and disease. Plenum Press, New York, p 59Google Scholar
  2. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  3. Cruz N, Antonio S, Anda R de, Gosset G, Bolivar F (1990) Preparative isolation by high performance liquid chromatography of human insulin B chain produced in Escherichia coli. J Liq Chromatogr 13:1517–1528Google Scholar
  4. Cruz N, López M, Estrada G, Alvarado X, Anda R de, Balbás P, Gosset G, Bolivar F (1992) Preparative isolation of recombinant human insulin A chain by ion exchange chromatography. J Liq Chromatogr 15:2311–2324Google Scholar
  5. Cutayar JM, Poillon D (1989) High cell density culture of E. coli in a fed-batch system with dissolved oxygen as substrate feed indicator. Biotechnol Lett 11:155–160Google Scholar
  6. Fass R, Clem TR, Shiloach J (1989) Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli. Appl Environ Microbiol 55:1305–1307Google Scholar
  7. Flores N, Anda R de, Guereca L, Cruz N, Antonio S, Balbás P, Bolivar F, Valle F (1986) A new expression vector for the production of fused proteins in Escherichia coli. Appl Microbiol Biotechnol 25:267–270Google Scholar
  8. Fu J, Togna AP, Shuler ML, Wilson DB (1992) Escherichia coli host cell modifications in continuous culture affecting heterologous protein overproduction: a population dynamics study. Biotechnol Prog 8:340–346Google Scholar
  9. Gleiser IE, Bauer S (1981) Growth of E. coli w to high cell concentration by oxygen level linked control of carbon source concentration. Biotechnol Bioeng 23:1015–1021Google Scholar
  10. Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD (1979) Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci 76:106–110.Google Scholar
  11. Koizumi J, Monden Y, Aiba S (1985) Effects of temperature and dilution rate on the copy number of recombinant plasmid in continuous culture of Bacillus stearothermophilus (pLP11). Biotechnol Bioeng 27:721–728Google Scholar
  12. Lee J, Youn-Hee C, Shin-Kwon K, Hyung-Hwan P, Ik-Boo K (1989) Production of human leukocyte interferon in Escherichia coli by control of growth rate in fed-batch fermentation. Biotechnol Lett 2:695–698Google Scholar
  13. Lee YL, Chang HN (1990) High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor. Biotechnol Bioeng 36:330–337Google Scholar
  14. Lowry OH, Rosebrough NH, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  15. Mizukami T, Oka T, Itoh S (1986) Production of active human interferon in E. coli II. optimal condition for induced synthesis by 3,B-indoleacrylic acid. Biotechnol Lett 8:611–614Google Scholar
  16. Paalme T, Tiisma K, Kahru A, Vanatalu K, Vilu R (1989) Glucose-limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate. Biotechnol Bioeng 35:312–319Google Scholar
  17. Pierce J, Gutteridge S (1985) Large-scale preparation of ribulose-bisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability. Appl Environ Microbiol 49:1094–1100Google Scholar
  18. Seo JH, Bailey JE (1985) Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol Bioeng 27:1668–1674Google Scholar
  19. Seo JH, Bailey JE (1986) Continuous cultivation of recombinant Escherichia coli: existence of an optimum dilution rate for maximum plasmid and gene product concentration. Biotechnol Bioeng 28:1590–1594Google Scholar
  20. Shiloach J, Bauer S (1975) High yield growth of E. coli. Biotechnol Bioeng 17:227–239Google Scholar
  21. Thompson BG, Kole M, Gerson DF (1985) Control of ammonium concentration in Escherichia coli fermentations. Biotechnol Bioeng 27:818–824Google Scholar
  22. Yansura DG, Henner DJ (1990) Use of Escherichia coli trp promoter for direct expression of proteins. Methods Enzymol 185:54–60Google Scholar
  23. Zabriskie DW, Arcuri EJ (1986) Factors influencing productivity of fermentations employing recombinant microorganisms. Enzyme Microb Technol 8:706–717Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • G. Gosset
    • 1
  • R. de Anda
    • 1
  • N. Cruz
    • 1
  • A. Martínez
    • 1
  • R. Quintero
    • 1
  • F. Bolivar
    • 1
  1. 1.Departamentos de Biología Molecular y Bioingeniería, Instituto de BiotechnologíaUniversidad Nacional Autónoma de México (UNAM)Cuernavaca, MorelosMexico

Personalised recommendations