Expression of human N-myristoyltransferase inEscherichia coli. Comparison with N-myristoyltransferases expressed in different tissues |
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Authors: | Rajala V S Raju Raju S S Datla Rajendra K Sharma |
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Institution: | (1) Department of Pathology and Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Royal University Hospital, S7N 4H4 Saskatoon, Saskatchewan;(2) Plant Biotechnology Institute, National Research Council, 110 Gymnasium place, S7N OW9 Saskatchewan, Saskatoon, Canada |
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Abstract: | 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 NIP711 a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. TheE. coli expressed hNMT was homogeneous and showed enzyme activity.Abbreviations NMT
N-myristoyl CoA:protein N-myristoyltransferase
- NIP71
71 kDa heat stable membrane bound N-myristoyltransferase inhibitor protein
- hNMT
human NMT
- DTNB
N-5,5dithiobis (2-nitrobenzoic acid)
- FPLC
fast protein liquid chromatography
- IPTG
isopropyl -D-thiogalactopyranoside
- cDNA
complementarydeoxyribonucleic acid
- SDS
sodium dodecyl sulfate
- PMSF
phenylmethylsulfonyl fluoride |
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Keywords: | myristoylation myristoyltransferase myristoyl CoA inhibition activation purification |
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