A silent mutation made possible efficient production of active human Frk tyrosine kinase in Escherichia coli.
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 mono-phosphorylated 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.
Yang
Xiaoyan
X
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan.
Kinoshita
Takayoshi
T
Gouda
Masaki
M
Yokota
Koichi
K
Tada
Toshiji
T
D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D009363:Neoplasm Proteins; D011505:Protein-Tyrosine Kinases; C092830:FRK protein, human