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Mar. Drugs 2015, 13(8), 5492-5507; doi:10.3390/md13085492

High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug

1
South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
2
Guangzhou Institute of Microbiology, Guangzhou 510663, China
3
Department of Pediatrics, The First Affiliated Hospital, Sun Yat-sen University, 58, Zhong Shan Second Road, Guangzhou 510080, China
*
Authors to whom correspondence should be addressed.
Academic Editors: Peter Proksch and Keith B. Glaser
Received: 7 May 2015 / Revised: 11 July 2015 / Accepted: 12 August 2015 / Published: 21 August 2015
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Abstract

l-Methionine γ-lyase (MGL), a pyridoxal 5′-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at −35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug. View Full-Text
Keywords: l-Methionine γ-lyase; purification; production; enzyme; leukemia; anti-leukemic drug; cell proliferation; apoptosis l-Methionine γ-lyase; purification; production; enzyme; leukemia; anti-leukemic drug; cell proliferation; apoptosis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Huang, K.-Y.; Hu, H.-Y.; Tang, Y.-L.; Xia, F.-G.; Luo, X.-Q.; Liu, J.-Z. High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug. Mar. Drugs 2015, 13, 5492-5507.

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