Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli.
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 degrees C, heat shock of the culture from 37 to 42 degrees C and from 37 to 45 degrees 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 degrees 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.
Kube
Jürgen
J
Bioprozess- und Bioverfahrenstechnik, Technische Universität Hamburg-Harburg, Denickestr. 15, 21071 Hamburg, Germany. j.kube@tuhh.de
Brokamp
Christian
C
Machielsen
Ronnie
R
van der Oost
John
J
Märkl
Herbert
H
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics*; D018533:Biomass; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature
D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase