Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src.

ABSTRACT: Site-directed mutagenesis is a traditional approach for structure-function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96-well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a pr

SUBMITTER: Cui Y 

PROVIDER: S-EPMC6371218 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature