An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system
2021
Emiko Kinoshita-Kikuta | Momoka Yoshimoto | Marina Yano | Eiji Kinoshita | Tohru Koike
ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.
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