High-level expression and bulk crystallization of recombinant l-methionine γ-lyase, an anticancer agent

Abstract

L-Methionine gamma-lyase is a pyridoxal 5'-phosphate-dependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.

The Role of Cysteine 116 in the Active Site of the Antitumor EnzymeL-Methionine γ-Lyase fromPseudomonas putida

“…The culture broth was transferred to 1.6 liter of fermentation medium (1.2% bacto tryptone, 2.4% bacto yeast extract, 2.0% glycerol, 1.25% K 2 HPO 4 , 0.23% KH 2 PO 4 , 0.05% polypropylene glycol no. 2000, and 10 mg/ml, tetracycline hydrochloride, pH 7.4) and cultivated at 28 C for 18 h with shaking. For expression of the recombinant MGL Pp, isopropyl--D-thiogalactopyranoside was added at a final concentration of 1 mM after cultivation for 4 h. The concentrated cell suspension was diluted with 50 mM potassium phosphate buffer (pH 7.2) containing 1 mM EDTA, 0.5 mM PLP, 0.01% dithiothreitol, and 1 mM PMSF.…”

Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene

“…Thus, depletion of L-methionine is effective for cancer therapy. The therapeutically utility of the enzyme for depleting L-methionine has been reported (2). The enzyme has been found to be an effective anti-tumor agent in vitro and in vivo and to be of potential value in the treatment of Parkinson's disease, arteriosclerosis, aging and obesity (9).…”

“…The antitumor efficacy of MGL, which is based on the greater L-methionine dependency of cancer cells for growth compared to normal cells has been investigated (3). Many human cancer cell lines and primary tumors have an absolute requirement for L-methionine, an essential amino acid (2). In vitro studies have demonstrated that several murine and human tumor cell lines need exogenous methionine to survive and proliferate (12).…”

“…Thus, depletion of L-methionine is effective for cancer therapy (13). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and become arrested in the late-S/G2 phase of the cell cycle (2,3,12,14). Cell cycle arrested and death can be reversed by addition of exogenous methionine (12).…”

“…These characteristics have resulted in the development of many enzyme drugs for a wide range of disorders (1). L-Methionine γ-lyase (MGL; EC 4.4.1.11) is a pyridoxal 5'-phosphate (PLP)-dependent multifunctional enzyme which catalyzes α, γ-elimination and γ-replacement of L-methionine and its derivatives and also α, β-elimination and β-replacement of S-substituted L-cysteines (2)(3)(4). This activity was first found using dried cells of E. coli and Proteus vulgaris (4).…”