EXPRESSION OF HUMAN N-MYRISTOYLTRANSFERASE IN ESCHERICHIA-COLI - COMPARISON WITH N-MYRISTOYLTRANSFERASES EXPRESSED IN DIFFERENT TISSUES

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukary otic proteins. Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the co mbination of ammonium sulfate precipitation, chromatography on SP-Seph arose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polya crylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp )(4)-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa wit hout loss of catalytic activity. The hNMT activity could be greatly ac tivated severalfold with the use of Tris, SDS, ethanol and acetonitril e. The catalytic activity of hNMT was potently inhibited in a concentr ation dependent manner by NIP71, a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. The E. coli expressed hNMT was homogeneous and showed enzyme activity.