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 eukaryotic proteins. Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose 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-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)4-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration 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.
Raju
R V
RV
Department of Pathology and Saskatoon Cancer Centre, University of Saskatchewan, Canada.
Datla
R S
RS
Sharma
R K
RK
D000217:Acyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004765:Enteropeptidase; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012997:Solvents
D011994:Recombinant Proteins; D012997:Solvents; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D004765:Enteropeptidase