Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation.
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 degrees 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 degrees 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.
Koma
Daisuke
D
Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba, 292-0812, Japan. koma-daisuke@nite.go.jp
Sawai
Toshiya
T
Harayama
Shigeaki
S
Kino
Kuniki
K
D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D043902:Pyrococcus horikoshii / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D000637:Transaminases / Q000235:genetics*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000637:Transaminases