Advertisement

Applied Microbiology and Biotechnology

, Volume 31, Issue 2, pp 158–162 | Cite as

Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

  • Julian A. Chesshyre
  • Alan R. Hipkiss
Applied Genetics and Regulation

Summary

The accumulation of interferon (IFN) α-2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon α-2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN α-2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t1/2 values for IFN α-2 decreased with increasing incubation temperature. Pulse-chase studies using [35S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN α-2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN α-2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.

Keywords

Sodium Escherichia Coli Electrophoresis Transition Temperature Interferon 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Boss MA, Kenton JH, Wood CR, Emtage JS (1984) Assembly of functional antibodies from immunoglobin heavy and light chains synthesized in E. coli. Nucleic Acids Res 12:3792–3806Google Scholar
  2. Carrier MJ, Nugent ME, Tacon WCA, Primrose SB (1983) High expression of cloned genes in E. coli and its consequences. Trends Biotechnol 1:109–113Google Scholar
  3. Chesshyre JA, Carr AJ, Hipkiss AR (1987) Proteolysis in the obligate methylotroph Methylophilus methylotrophus. Arch Microbiol 147:394–398Google Scholar
  4. Edge MD, Camble R, Moore VE, Hockney RC, Carr FJ, Fitton J (1986) Interferon analogues from synthetic genes: an approach to protein structure-activity studies. In: Gresser I (ed) Interferon 7. Academic Press, London, pp 1–47Google Scholar
  5. Goff S, Goldberg AL (1985) Production of abnormal proteins in Escherichia coli stimulates transcription of lon and other heat shock genes. Cell 41:589–595Google Scholar
  6. Gow AJ, Hipkiss AR (1989) Genetic and physiological modulation of interferon accumulation in Escherichia coli. Biochem Soc Trans 17:180–181.Google Scholar
  7. Gross G, Mayr U, Straber D, Westphil W, Kacher M, Frank R, Blocker H (1985) Stabilization of human interferon β synthesis in Escherichia coli by zinc ions. Biochim Biophys Acta 825:207–213Google Scholar
  8. Hipkiss AR (1979) Inhibition of breakdown of canavanyl proteins in Escherichia coli by chloramphenicol. FEMS Microbiol Lett 6:349–353Google Scholar
  9. Kemshead JT, Hipkiss AR (1974) Degradation of abnormal proteins in Escherichia coli: relative susceptibility of canavanyl proteins and puromycin peptides to proteolysis in vitro. Eur J Biochem 45:535–540Google Scholar
  10. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head protein of bacteriophage T4. Nature 227:680–685Google Scholar
  11. Lin S, Zabin I (1972) β-Galactosidase: rates of synthesis and degradation of incomplete proteins. J Biol Chem 247:2205–2212Google Scholar
  12. Miller JH (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, pp 431–432Google Scholar
  13. Mitraki A, Belton JM, Desmadril M, Yon JM (1987) Quasireversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanine hydrochloride. Eur J Biochem 163:29–34Google Scholar
  14. Neuhoff V, Stamm R, Eibl H (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6:427–448Google Scholar
  15. Privalov PL (1979) Stability of proteins. Adv Protein Chem 247:2205–2212Google Scholar
  16. Schein CH, Noteborn MNH (1988) Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Biotechnology 6:291–294Google Scholar
  17. Shen SH (1984) Multiple joined genes prevent product degradation in Escherichia coli. Proc Natl Acad Sci USA 81:4627–4631Google Scholar
  18. Shiroza T, Nakazawa K, Tashiro N, Yamane K, Yanagi K, Yamasaki M, Tamura G, Saito H, Kawade Y, Taniguchi T (1985) Synthesis and secretion of biologically active interferon-β using a Bacillus subtilis α-amylase secretion vector. Gene 34:1–8Google Scholar
  19. Snow A, Hipkiss AR (1987) Stability of urogastrone and some fusion derivatives and their induction of stress proteins in Escherichia coli. Biochem Soc Trans 15:965–966Google Scholar
  20. Swamy KH, Goldberg AL (1981) Escherichia coli contains eight soluble proteolytic activities, one being ATP-dependent. Nature 292:652–654Google Scholar
  21. Thatcher DR, Panayotatos N (1986) Purification of recombinant human IFN α-2. In: Pestke S (ed), Colewick SP, Kaplan SP (series eds) Methods in enzymology, vol 119, Academic Press, London, pp 166–177Google Scholar
  22. Wetzel A, Johnston PD, Czarniecki CW (1983) Roles of the disulphide bonds in a human alpha interferon. In: de Maeyer E, Schellekens H (eds) The biology of the interferon system. Elsevier, Amsterdam, pp 101–112Google Scholar
  23. Windass J, Newton CR, De Maeyer-Guignard J, Moore VE, Markham AF, Edge MD (1982) The construction of a synthetic Escherichia coli trp promotor and its use in the expression of a synthetic interferon gene. Nucl Acid Res 10:6639–6657Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Julian A. Chesshyre
    • 1
  • Alan R. Hipkiss
    • 1
  1. 1.Department of Biochemistry, King's College LondonUniversity of LondonLondonUnited Kingdom

Personalised recommendations