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

Marine Drugs

Kui-Ying Huang²  Hai-Yan Hu³  Yan-Lai Tang¹  Feng-Geng Xia³  Xue-Qun Luo¹  Jian-Zhong Liu²  Peter Proksch⁴ 

[1] Department of Pediatrics, The First Affiliated Hospital, Sun Yat-sen University, 58, Zhong Shan Second Road, Guangzhou 510080, China; E-Mail:;South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; E-Mail:;Guangzhou Institute of Microbiology, Guangzhou 510663, China; E-Mails:;South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; E-Mail

关键词: l-Methionine γ-lyase;    purification;    production;    enzyme;    leukemia;    anti-leukemic drug;    cell proliferation;    apoptosis;   

DOI  :  10.3390/md13085492

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.