A Silent Mutation Made Possible Efficient Production of Active Human Frk Tyrosine Kinase in Escherichia coli

作者：Yang Xiaoyan; Kinoshita Takayoshi^*; Gouda Masaki; Yokota Koichi; Tada Toshiji

来源：Bioscience Biotechnology and Biochemistry, 2010, 74(1): 125-128.

DOI：10.1271/bbb.90648

• silent mutant
• Fyn-related kinase (Frk)
• bacterial expression

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors. The excess activity of Frk on beta-cells has a crucial role in type-I diabetes. A silent mutation at Ile229 conferred a bacterial expression system on the kinase domains of Frk, which allowed for the quick expression and purification of one unphosphorylated and two monophosphorylated kinase domains. The C-terminal catalytic segment of the human Frk kinase conjugating hexahistidine purification tag (His-tag) was expressed in Escherichia coli. After first-step purification utilizing the His-tag, an anion-exchange chromatogram yielded three major peaks that had distinguishable phosphorylation characteristics as judged by Western blot analysis and measurement of kinase activity. This result of active protein production should promote drug discovery studies, including highthrough-put screening and structure-based drug design.

• 出版日期2010-1