An improved method and cost effective strategy for soluble expression and purification of human N-myristoyltransferase 1 in E. coli.

Abstract

N-myristoyltransferase (NMT) is an indispensible enzyme, which exists as two isoforms (NMT1 and NMT2) in humans and has proven roles in development of cancerous states. It is thus a target for novel anti-cancer drug design, but understanding of the biochemical and functional differences of these isozymes is not fully deciphered. A soluble expression under the T7 promoter for human NMT1 was achieved in E. coli BL21 (DE3) cells, devoid of any isopropyl β-D-1-thiogalactopyranoside-based induction. The identity of expressed protein was confirmed by matrix-assisted laser desorption ionization mass spectrometry peptide-fingerprint analysis and a two-step purification protocol yielded homogeneous enzyme. The intact mass of the purified protein was verified by electrospray ionization mass spectrometry and found to be in agreement with the theoretical mass (48.141 vs. 48.140 kDa). The fluorescence spectrophotometric analyses of the ligand binding and enzyme activity demonstrated that the recombinant form is functional. The yield of purified protein was ~8-10 mg/L culture (batch to batch variation) with a specific activity value of 18,500 ± 513 U/mg of protein under the experimental conditions used. The final verification of the myristoylation was demonstrated by mass spectrometry analysis of reaction product. The described approach could be readily adapted for production of human NMT1, with high yields of pure enzyme preparations, which should aid in downstream applications involving inhibitor design and structure-function studies of NMT's.

• Authors: Sujeet Kumar, Rajendra K Sharma
• Journal: Molecular and cellular biochemistry (Mol Cell Biochem) Vol. 392 Issue 1-2 Pg. 175-86 (Jul 2014) ISSN: 1573-4919 [Electronic] Netherlands
• PMID: 24668448 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA Primers
• Recombinant Proteins
• Acyltransferases
• glycylpeptide N-tetradecanoyltransferase

Topics

• Acyltransferases (chemistry, genetics, isolation & purification)
• Amino Acid Sequence
• Base Sequence
• Cost-Benefit Analysis
• DNA Primers
• Escherichia coli (genetics)
• Humans
• Mass Spectrometry
• Molecular Sequence Data
• Polymerase Chain Reaction
• Recombinant Proteins (chemistry, genetics, isolation & purification)
• Spectrometry, Fluorescence