Expression, purification and in vitro N-myristoylation of human Src N-terminal region

The DNA fragment encoding N-terminal region of human c-Src was amplified from Caco-2 cell total RNA by RT-PCR and cloned into vector pMFHT to obtain His-tag fusion expression plasmid pMF-SrcHT, which was based on T7 expression system. The fusion protein SrcHT was highly expressed in E.coli BL21(DE3) harboring the pMF-SrcHT and purified from bacterial lysate by Ni-IDA affinity chromatography. The assays using [(3)H]-labeled substrate demonstrate that the purified fusion protein SrcHT can be effectively N-myristoylated by recombinant human myristoyl-CoA: protein N-myristoyltransferase (NMT) in vitro. This work is a basis for further biochemical studies and development of new anti-cancer chemotherapeutic drugs based on specific inhibition of N-myristoylation of human Src.