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.
Ma
Han-Hui
HH
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Yang
Li
L
Li
Bo-Liang
BL
D000595:Amino Acid Sequence; D001483:Base Sequence; D018938:Caco-2 Cells; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D015688:Oncogene Protein pp60(v-src)