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Expression of human N-myristoyltransferase inEscherichia coli. Comparison with N-myristoyltransferases expressed in different tissues

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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.

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Abbreviations

NMT:

N-myristoyl CoA:protein N-myristoyltransferase

NIP71 :

71 kDa heat stable membrane bound N-myristoyltransferase inhibitor protein

hNMT:

human NMT

DTNB:

N-5,5′dithiobis (2-nitrobenzoic acid)

FPLC:

fast protein liquid chromatography

IPTG:

isopropyl β-D-thiogalactopyranoside

cDNA:

complementarydeoxyribonucleic acid

SDS:

sodium dodecyl sulfate

PMSF:

phenylmethylsulfonyl fluoride

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Authors and Affiliations

  1. Department of Pathology and Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Royal University Hospital, S7N 4H4, Saskatoon, Saskatchewan

    Rajala V. S. Raju & Rajendra K. Sharma

  2. Plant Biotechnology Institute, National Research Council, 110 Gymnasium place, S7N OW9, Saskatchewan, Saskatoon, Canada

    Raju S. S. Datla

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  1. Rajala V. S. Raju
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  2. Raju S. S. Datla
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Raju, R.V.S., Datla, R.S.S. & Sharma, R.K. Expression of human N-myristoyltransferase inEscherichia coli. Comparison with N-myristoyltransferases expressed in different tissues. Mol Cell Biochem 155, 69–76 (1996). https://doi.org/10.1007/BF00714335

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  • DOI: https://doi.org/10.1007/BF00714335

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