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

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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.

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Acknowledgments

This research was supported by the Canadian Breast Cancer Foundation. The authors are thankful to Dr. Linda Chelico, University of Saskatchewan for the use of BioLogic DuoFlow chromatographic system. Dr. Sreejit Paramaeswaran is thanked for critically reading the manuscript.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, SK, S7N 0W8, Canada

    Sujeet Kumar & Rajendra K. Sharma

  2. Cancer Cluster, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada

    Sujeet Kumar & Rajendra K. Sharma

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  1. Sujeet Kumar
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Correspondence to Rajendra K. Sharma.

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11010_2014_2029_MOESM1_ESM.tif

Supplementary material 1 Supplementary figure 1 as TIFF image. Fig. S1 Section of ESI mass spectrum of purified hNMT1s. The experimentally determined molecular weight of 48141 Da is in accordance with the theoretical value of 48140.81 Da

11010_2014_2029_MOESM2_ESM.tif

Supplementary material 2 Supplementary figure 2 as TIFF image. Fig. S2 Structural model of the human N-myristoyltransferase representing the spatial location of the catalytic domain Trp 40 in the vicinity of bound ligand myristoyl-CoA. The image was generated from the PDB file 3IU1 with VMD (ver 1.8.7) [A]. [A] Humphrey W, Dalke A, Schulten K: VMD - Visual Molecular Dynamics. J Mol Graph 1996, 14:33-38

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Kumar, S., Sharma, R.K. An improved method and cost effective strategy for soluble expression and purification of human N-myristoyltransferase 1 in E. coli . Mol Cell Biochem 392, 175–186 (2014). https://doi.org/10.1007/s11010-014-2029-z

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