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Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation

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Abstract

Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 °C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 °C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.

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Acknowledgements

This study was supported in part by the 21C-COE Program “Practical Nano-Chemistry” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and by a Waseda University Grant for Special Research Projects (2005B-142). We are grateful to Dr. Katsumi Isono for the critical review and helpful discussions.

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Correspondence to Daisuke Koma.

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Koma, D., Sawai, T., Harayama, S. et al. Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation. Appl Microbiol Biotechnol 73, 172–180 (2006). https://doi.org/10.1007/s00253-006-0448-9

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Keywords

  • Soluble Protein
  • Crude Enzyme
  • Aminotransferase Activity
  • Folding Rate
  • Heterologous Gene Expression