Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.

ABSTRACT: A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (k_cat/K_M) of purified ABL kinase for the lead peptide (125 s^-1 μM^-1) is similar to that of the starting peptide (103 s^-1 μM^-1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, th

SUBMITTER: Proctor A 

PROVIDER: S-EPMC5111365 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature