High-Level Expression, Purification and Large-Scale Production of L-Methionine gamma-Lyase from Idiomarina as a Novel Anti-Leukemic Drug
Huang, Kui-Ying; Hu, Hai-Yan; Tang, Yan-Lai; Xia, Feng-Geng; Luo, Xue-Qun; Liu, Jian-Zhong; qiushuixian2004@163.com; huhaiyansysu@163.com; tangyanlai@126.com; xiafenggeng@163.com; L-xuequn@126.com; lssljz@mail.sysu.edu.cn
2015
发表期刊MARINE DRUGS
卷号13期号:8页码:5492-5507
摘要l-Methionine -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 degrees 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.
部门归属[Huang, Kui-Ying; Liu, Jian-Zhong] Sun Yat Sen Univ, Sch Life Sci, South China Sea Bioresource Exploitat & Utilizat, Guangzhou 510275, Guangdong, Peoples R China; [Huang, Kui-Ying; Hu, Hai-Yan; Xia, Feng-Geng] Guangzhou Inst Microbiol, Guangzhou 510663, Guangdong, Peoples R China; [Tang, Yan-Lai; Luo, Xue-Qun] Sun Yat Sen Univ, Affiliated Hosp 1, Dept Pediat, Guangzhou 510080, Guangdong, Peoples R China ; LTO
关键词L-methionine -lyase Purification Production Enzyme Leukemia Anti-leukemic Drug Cell Proliferation Apoptosis
学科领域Pharmacology & Pharmacy
文献类型期刊论文
条目标识符http://ir.scsio.ac.cn/handle/344004/14934
专题热带海洋环境国家重点实验室(LTO)
通讯作者qiushuixian2004@163.com; huhaiyansysu@163.com; tangyanlai@126.com; xiafenggeng@163.com; L-xuequn@126.com; lssljz@mail.sysu.edu.cn
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Huang, Kui-Ying,Hu, Hai-Yan,Tang, Yan-Lai,et al. High-Level Expression, Purification and Large-Scale Production of L-Methionine gamma-Lyase from Idiomarina as a Novel Anti-Leukemic Drug[J]. MARINE DRUGS,2015,13(8):5492-5507.
APA Huang, Kui-Ying.,Hu, Hai-Yan.,Tang, Yan-Lai.,Xia, Feng-Geng.,Luo, Xue-Qun.,...&lssljz@mail.sysu.edu.cn.(2015).High-Level Expression, Purification and Large-Scale Production of L-Methionine gamma-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.MARINE DRUGS,13(8),5492-5507.
MLA Huang, Kui-Ying,et al."High-Level Expression, Purification and Large-Scale Production of L-Methionine gamma-Lyase from Idiomarina as a Novel Anti-Leukemic Drug".MARINE DRUGS 13.8(2015):5492-5507.
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