An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system

METHOD SUMMARY This article describes an assay of tyrosine protein kinase ABL activity using an Escherichia coli protein expression system. Human tyrosine kinase ABL and its peptide substrate (Abltide) tagged with glutathione S-transferase were co-expressed in E. coli, and tyrosine phosphorylation of the substrate was detected using Phos-tag SDS-PAGE, followed by western blotting with anti-glutathione S-transferase antibody. This method was used for an assay of the kinase activity of wild-type ABL and five of its kinase domain mutants (Y253F, E255K, T315I, M351T and H396P) that are associated with imatinib resistance in patients with chronic myeloid leukemia. 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.

Automated summary

This article discusses an assay of tyrosine protein kinase ABL activity using an Escherichia coli protein expression system. The study involves co-expressing human tyrosine kinase ABL and its substrate in E. coli and detecting tyrosine phosphorylation using Phos-tag SDS-PAGE. Results show different kinase activity levels in mutant forms of ABL compared to the wild-type, indicating a correlation between phosphorylation states and enzymatic activity.