Extremophiles

, Volume 10, Issue 3, pp 221–227 | Cite as

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

  • Jürgen Kube
  • Christian Brokamp
  • Ronnie Machielsen
  • John van der Oost
  • Herbert Märkl
Original Paper

Abstract

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.

Keywords

Protein production Thermoactive ADH Pyrococcus furiosus Escherichia coli Overexpression 

Notes

Acknowledgements

The work was sponsored by the EU fifth Framework program PYRED (QLTR-2000-01676).

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jürgen Kube
    • 1
  • Christian Brokamp
    • 1
  • Ronnie Machielsen
    • 2
  • John van der Oost
    • 2
  • Herbert Märkl
    • 1
  1. 1.Bioprozess- und BioverfahrenstechnikTechnische Universität Hamburg-HarburgHamburgGermany
  2. 2.Laboratory of MicrobiologyWageningen UniversityCT WageningenThe Netherlands

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