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Open AccessArticle

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

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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
Mar. Drugs 2015, 13(8), 5492-5507; https://doi.org/10.3390/md13085492
Received: 7 May 2015 / Revised: 11 July 2015 / Accepted: 12 August 2015 / Published: 21 August 2015
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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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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