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. 1990 Mar;56(3):640–645. doi: 10.1128/aem.56.3.640-645.1990

Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12.

H L MacDonald 1, J O Neway 1
PMCID: PMC183398  PMID: 2180368

Abstract

We examined the ability of transformed Escherichia coli cells in fermentor cultures to accumulate interleukin-2 (IL-2) intracellularly under temperature-regulated control of the phage lambda pL promoter. Induction of expression was undertaken at different culture optical densities, and specific IL-2 accumulation was found to decrease with increasing cell density at induction. Induction at higher culture optical densities was also accompanied by decreased growth during induction and increased acetate accumulation in the culture medium. Experiments were undertaken to study the effect of replacing spent medium by perfusion with fresh medium both before induction and during IL-2 expression at high cell density. Improved IL-2 expression was seen only when perfusion was continued past 1.6 h after the start of induction, and it was accompanied by a significant reduction in acetate buildup. Further improvements were not seen when perfusion was continued beyond hour 3 of induction. Replenishing medium components and decreasing the concentration of diffusible inhibitors before induction did not alleviate acetate buildup, growth limitation, or limitation of IL-2 synthesis. These results suggested that accumulation of diffusible inhibitors such as acetate during induction may be a significant factor limiting IL-2 expression in high-density cultures, but other factors intrinsic to the organism or the protein also played a major role.

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Selected References

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

  1. Baracchini E., Bremer H. Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp. J Biol Chem. 1988 Feb 25;263(6):2597–2602. [PubMed] [Google Scholar]
  2. Bauer S., Shiloach J. Maximal exponential growth rate and yield of E. coli obtainable in a bench-scale fermentor. Biotechnol Bioeng. 1974 Jul;16(7):933–941. doi: 10.1002/bit.260160707. [DOI] [PubMed] [Google Scholar]
  3. Brown T. D., Jones-Mortimer M. C., Kornberg H. L. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol. 1977 Oct;102(2):327–336. doi: 10.1099/00221287-102-2-327. [DOI] [PubMed] [Google Scholar]
  4. Caulcott C. A., Dunn A., Robertson H. A., Cooper N. S., Brown M. E., Rhodes P. M. Investigation of the effect of growth environment on the stability of low-copy-number plasmids in Escherichia coli. J Gen Microbiol. 1987 Jul;133(7):1881–1889. doi: 10.1099/00221287-133-7-1881. [DOI] [PubMed] [Google Scholar]
  5. Chang Y. Y., Cronan J. E., Jr Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J Bacteriol. 1983 May;154(2):756–762. doi: 10.1128/jb.154.2.756-762.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang Y. Y., Cronan J. E., Jr Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase. J Bacteriol. 1982 Sep;151(3):1279–1289. doi: 10.1128/jb.151.3.1279-1289.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goff S. A., Goldberg A. L. An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli. J Biol Chem. 1987 Apr 5;262(10):4508–4515. [PubMed] [Google Scholar]
  8. Grossman A. D., Taylor W. E., Burton Z. F., Burgess R. R., Gross C. A. Stringent response in Escherichia coli induces expression of heat shock proteins. J Mol Biol. 1985 Nov 20;186(2):357–365. doi: 10.1016/0022-2836(85)90110-x. [DOI] [PubMed] [Google Scholar]
  9. Harrison D. E., Pirt S. J. The influence of dissolved oxygen concentration on the respiration and glucose metabolism of Klebsiella aerogenes during growth. J Gen Microbiol. 1967 Feb;46(2):193–211. doi: 10.1099/00221287-46-2-193. [DOI] [PubMed] [Google Scholar]
  10. Jones I. M., Primrose S. B., Robinson A., Ellwood D. C. Maintenance of some ColE1-type plasmids in chemostat culture. Mol Gen Genet. 1980;180(3):579–584. doi: 10.1007/BF00268063. [DOI] [PubMed] [Google Scholar]
  11. Jung G., Denèfle P., Becquart J., Mayaux J. F. High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1 beta. Ann Inst Pasteur Microbiol. 1988 Jan-Feb;139(1):129–146. doi: 10.1016/0769-2609(88)90100-7. [DOI] [PubMed] [Google Scholar]
  12. Kwan H. S., Chui H. W., Wong K. K. ack::Mu d1-8 (Apr lac) operon fusions of Salmonella typhimurium LT2. Mol Gen Genet. 1988 Jan;211(1):183–185. doi: 10.1007/BF00338411. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Pascal M. C., Chippaux M., Abou-Jaoudé A., Blaschkowski H. P., Knappe J. Mutants of Escherichia coli K12 with defects in anaerobic pyruvate metabolism. J Gen Microbiol. 1981 May;124(1):35–42. doi: 10.1099/00221287-124-1-35. [DOI] [PubMed] [Google Scholar]
  15. Pedersen S., Bloch P. L., Reeh S., Neidhardt F. C. Patterns of protein synthesis in E. coli: a catalog of the amount of 140 individual proteins at different growth rates. Cell. 1978 May;14(1):179–190. doi: 10.1016/0092-8674(78)90312-4. [DOI] [PubMed] [Google Scholar]
  16. Phillips T. A., VanBogelen R. A., Neidhardt F. C. lon gene product of Escherichia coli is a heat-shock protein. J Bacteriol. 1984 Jul;159(1):283–287. doi: 10.1128/jb.159.1.283-287.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sarubbi E., Rudd K. E., Cashel M. Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli. Mol Gen Genet. 1988 Aug;213(2-3):214–222. doi: 10.1007/BF00339584. [DOI] [PubMed] [Google Scholar]
  18. St John A. C., Goldberg A. L. Effects of reduced energy production on protein degradation, guanosine tetraphosphate, and RNA synthesis in Escherichia coli. J Biol Chem. 1978 Apr 25;253(8):2705–2711. [PubMed] [Google Scholar]
  19. Wang A. M., Creasey A. A., Ladner M. B., Lin L. S., Strickler J., Van Arsdell J. N., Yamamoto R., Mark D. F. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science. 1985 Apr 12;228(4696):149–154. doi: 10.1126/science.3856324. [DOI] [PubMed] [Google Scholar]
  20. 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]

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