Applied Microbiology and Biotechnology

, Volume 28, Issue 2, pp 170–175 | Cite as

Construction of a runaway vector and its use for a high-level expression of a cloned human superoxide dismutase gene

  • Tomio Morino
  • Makoto Morita
  • Kenji Seya
  • Yoshikazu Sukenaga
  • Kazuo Kato
  • Tsuneo Nakamura
Applied Genetics and Regulation

Summary

A runaway cloning vector pR3 from plasmid pBEU17 has been constructed by introducing three deletions (ΔI, ΔH and ΔEHi), multiple cloning sites and transcription terminator sequences. Introduction of the deletion ΔH restored a drastic plasmid amplification at high temperature (37° C). This suggested the presence of unknown sequences around the ΔH region which repressed runaway replication. To examine the usefulness of a pR3 runaway vector on the high-level expression of a cloned gene, a pRT8 plasmid containing a cloned human superoxide dismutase (SOD) gene in the pR3 was constructed. A host with tolerance to high copy numbers of pRT8 was selected, and the cultural conditions required for high-level expression were investigated. It was shown that the copy number of pRT8 increased in the stationary phase even at 30° C, and that cells carrying an excess number of plasmid did not support the runaway replication, yielding less SOD protein. SOD synthesis in the production culture lasted for about 24 h after the thermal shift, though viability was soon lost. The maximum yield was determined as approximately 20% of total cellular protein. This was 12-fold higher than that of a vector having the ColE1 plasmid replicon.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Burger KJ, Steinbauer J, Rollich G, Geobol W (1981) Copy number control and incompatibility of plasmid R1: identification of a protein that seemed to be involved in both process. Mol Gen Genet 182:44–52Google Scholar
  2. Boer HA de, Comstock LJ, Vasser M (1983) Thetac promoter; a functional hybrid derived fromtrp andlac promoters. Proc Natl Acad Sci USA 80:21–25Google Scholar
  3. Hallewell RA, Masiarz FR, Najarian RC, Puma JP, Quiroza MR, Randolph A, Sanchez-Pescador P, Scandella CJ, Smith B, Steimer KS, Mullenbach GT (1985) Human Cu/Zn superoxide dismutanse cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library. Nucleic Acids Res 13:2017–2034Google Scholar
  4. Hartman JR, Geller T, Yavin Z, Bartfeld D, Kanner D, Aviv H, Gorecki M (1986) High-level expression of enzymatically active human Cu/Zn superoxide dimutase inEscherichia coli. Proc Natl Acad Sci USA 83:7142–7146Google Scholar
  5. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 277:680–685Google Scholar
  6. Lewington J, Day MJ (1986) A rapid electrophoretic method for the measurement of plasmid copy number. Lett Appl Microbiol 3:109–112Google Scholar
  7. Nishimura N, Ito Y, Adachi T, Hirano K, Sugiura M, Sawai S (1982) Enzyme immunoassay of CuproZinc-superoxide dismutase in serum and urine. J Pharm Dyn 5:161–170Google Scholar
  8. Remaut E, Tsao H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene 22:103–113Google Scholar
  9. Sherman L, Dafini N, Lieman-Hurwitz J, Groner Y (1983) Nucleotide sequence and expression of human chromosome 21 encoded superoxide dismutase mRNA. Proc Natl Acad Sci USA 80:5465–5469Google Scholar
  10. Shoner BE, Hsiun HM, Belagaje RM, Mayne NG, Schoner RG (1984) Role of mRNA translational efficiency in bovine growth hormone expression inEscherichia coli. Proc Natl Acad Sci USA 81:5403–5407Google Scholar
  11. Simons G, Remaut E, Allet B, Devos R, Fiers W (1984) High level expression of human interferon gamma inEscherichia coli under the control of the Pl promoter of bacteriophage lambda. Gene 28:55–64Google Scholar
  12. Stougaar P, Moline S, Nordstrom K (1981) RNAs involved in copy number control and incompatibility of plasmid R1. Proc Natl Acad Sci USA 78:6008–6012Google Scholar
  13. Sukenaga Y, Morino T, Kato K, Nakamura T (1986) Molecular cloning and high level gene expression of superoxide dismutase and protein purification. Bioindustry (Tokyo) 3:961–970Google Scholar
  14. Uhlin BE, Nordstrom K (1978) A runaway replication mutant of plasmid R1drd-19. Mol Gen Genet 165:167–179Google Scholar
  15. Uhlin BE, Clark AJ (1981) Overproduction of theEscherichia coli recA protein without stimulation of its proteolytic activity. J Bacteriol 148:386–390Google Scholar
  16. Uhlin BE, Molin S, Gulstafsson P, Nordstrom K (1979) Plasmid with temperature-dependent copy number for amplification of cloned genes and their products. Gene 6:91–106Google Scholar
  17. Yasuda S, Takagi T (1983) Overproduction ofEscherichia coli replication proteins by the use of runaway replication plasmids. J Bacteriol 154:1153–1161Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Tomio Morino
    • 1
  • Makoto Morita
    • 1
  • Kenji Seya
    • 1
  • Yoshikazu Sukenaga
    • 1
  • Kazuo Kato
    • 1
  • Tsuneo Nakamura
    • 1
  1. 1.Research Laboratories, Pharmaceuticals GroupNippon Kayaku Co. Ltd.TokyoJapan

Personalised recommendations