Abstract
C-Src is a member of the non-receptor tyrosine kinase family and it is one of the major therapeutic targets due to its critical role in many cellular signaling pathways. The phosphorylation states of Tyrosine 416 and Tyrosine 527 in the catalytic domain of c-Src determine c-Src’s conformations and activities. Therefore an equilibrium exists between the species with different phosphorylation states on both Tyrosine residues. To investigate the population of each species at equilibrium, NMR is required. Unfortunatelyc-Src’s large size and low expression level poses a major obstacle. To resolve this a SortaseA-mediated method is adopted. This method allows for separate expression of the SH3SH2 domains and the catalytic domain of c-Src followed by a back-ligation, which makes the complete product. This back-ligated Src’sin vitro similarity to full-length Src is determined by Western blot, ATP-NADH coupled assay and NMR spectra. The insertion of the SortaseA motif LPETGG between Alanine 259 and Tryptophan 260 disrupts the interaction between the SH2-catalytic domain linker and the SH3 domain, which turns out to be critical for Src’s regulation. A new ligation scheme is proposed in which residue Threonine246 to Serine251 is substituted with the SortaseA motif. A full-length construct with the substitution is first acquired to determine whether it still interferes with the interaction between the linker and the SH3 domain. Current results of the ATP-NADH coupled assay and NMR spectra provide support for next-step back-ligation studies of this new construct.