title: High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.
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.
authors: South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China. qiushuixian2004@163.com.|Guangzhou Institute of Microbiology, Guangzhou 510663, China. qiushuixian2004@163.com.
authors: Kui-Ying
authors: KY
authors: Huang
authors: Guangzhou Institute of Microbiology, Guangzhou 510663, China. huhaiyansysu@163.com.
authors: Hai-Yan
authors: HY
authors: Hu
authors: Department of Pediatrics, The First Affiliated Hospital, Sun Yat-sen University, 58, Zhong Shan Second Road, Guangzhou 510080, China. tangyanlai@126.com.
authors: Yan-Lai
authors: YL
authors: Tang
authors: Guangzhou Institute of Microbiology, Guangzhou 510663, China. xiafenggeng@163.com.
authors: Feng-Geng
authors: FG
authors: Xia
authors: Department of Pediatrics, The First Affiliated Hospital, Sun Yat-sen University, 58, Zhong Shan Second Road, Guangzhou 510080, China. L-xuequn@126.com.
authors: Xue-Qun
authors: XQ
authors: Luo
authors: South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China. lssljz@mail.sysu.edu.cn.
authors: Jian-Zhong
authors: JZ
authors: Liu
mesh_terms: D034241:Alteromonadaceae / Q000737:chemistry / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000188:drug therapy*
chemical_list: D000970:Antineoplastic Agents; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase
keywords: anti-leukemic drug; apoptosis; cell proliferation; enzyme; l-Methionine γ-lyase; leukemia; production; purification