Overexpression of human N-myristoyltransferase utilizing a T7 polymerase gene expression system.
Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding human N-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 pp60src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS-polyacrylamide gel electrophoresis. Antibodies to Escherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The over-expressed hNMT was homogeneous and showed enzyme activity.
Raju
R V
RV
Department of Pathology, College of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Canada.
Datla
R S
RS
Sharma
R K
RK
D000217:Acyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000918:Antibody Specificity; D002847:Chromatography, Agarose; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis / Q000276:immunology; D015151:Immunoblotting; D010957:Plasmids; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011817:Rabbits; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D013862:Thiogalactosides / Q000378:metabolism
D007106:Immune Sera; D013501:Surface-Active Agents; D013862:Thiogalactosides; D012967:Sodium Dodecyl Sulfate; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D012321:DNA-Directed RNA Polymerases