Abstracts Category : Other

Progress towards mechanistic studies of Src tyrosine kinase using ligated protein constructs

by Sara Gelles-Watnick

Tyrosine kinase c-Src is a ubiquitously expressed proto-oncogene implicated in manycancers. The Kern Lab hopes to use NMR to study the regulatory and catalyticmechanisms of c-Src. Because of the large size of c-Src, a partially labeled version ofFull-length (FL) Src was required for NMR studies. To prepare the partially labelledprotein, part of c-Src was isotopically labelled and then ligated to the remainder of theprotein using the Sortase enzyme. Two variants of FL Src amenable to Sortase ligation(Back-ligated Src 1 and Back-ligated Src 2) were designed and generated by adding theSortase ligation motifs at different locations in the sequence. By a coupled peptidekinaseactivity assay, BL Src 1 and BL Src 2 were determined to have activity verysimilar to each other, and activity roughly similar to FL Src. Comparing the NMR spectraof BL Src 1 and BL Src 2 revealed that the BL Src 2 most closely mimics FL Src. Thus,BL Src 2 should be used in future NMR studies of the Src tyrosine kinase mechanism.