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