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

Kui-Ying Huang,Yan-Lai Tang,Xue-Qun Luo,Feng-Geng Xia,Hai-Yan Hu,Jian-Zhong Liu

doi:10.3390/md13085492

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.

Highlights

L-Methionine γ-lyase (MGL), a pyridoxal 5′-phosphate-dependent enzyme, catalyzes the γ-elimination of L-Methionine to generate α-ketobutyrate, methanethiol, and ammonia as well as the α,β-replacement and β-elimination of S-substituted L-cysteines [1]
We found that flavokawain B might be a possible therapeutic drug for patients with acute lymphoblastic leukemia (ALL)
We demonstrated the anti-leukemic effect of recombinant MGL (rMGL) in ALL and acute myeloid leukemia (AML) cell lines

Summary

Introduction

L-Methionine γ-lyase (MGL), a pyridoxal 5′-phosphate-dependent enzyme, catalyzes the γ-elimination of L-Methionine to generate α-ketobutyrate, methanethiol, and ammonia as well as the α,β-replacement and β-elimination of S-substituted L-cysteines [1]. To produce the enzyme on a large scale, a scale-up production process for the recombinant MGL (rMGL) was established, which contained a heat step, two diethyl-aminoethanol (DEAE) Sepharose. Fast Flow (FF) ion-exchanges, and one ActiClen Exox endotoxin-affinity chromatography column [5]. Another efficient production process was built-in by plasmid overexpression and cultivation and crystallization on an industrial scale [6]. Their processes could be used to produce rMGL on a large scale, the activity of the enzyme was still low

Methods

Results

Discussion

Conclusion