Overexpression of HumanN-Myristoyltransferase Utilizing a T7 Polymerase Gene Expression System

Myristoyl CoA:proteinN-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding humanN-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP–Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60^src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS–polyacrylamide gel electrophoresis. Antibodies toEscherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The overexpressed hNMT was homogeneous and showed enzyme activity.