High-level expression and bulk crystallization of recombinant L-methionine gamma-lyase, an anticancer agent.
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.
Takakura
Tomoaki
T
Discovery Research Laboratories, Shionogi & Co., Ltd., Amagasaki, Hyogo, Japan. tomoaki.takakura@shionogi.co.jp
Ito
Takaomi
T
Yagi
Shigeo
S
Notsu
Yoshihide
Y
Itakura
Takashi
T
Nakamura
Takumi
T
Inagaki
Kenji
K
Esaki
Nobuyoshi
N
Hoffman
Robert M
RM
Takimoto
Akio
A
D000964:Antimetabolites, Antineoplastic; D001709:Biotechnology / Q000379:methods; D013437:Carbon-Sulfur Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003460:Crystallization; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D000964:Antimetabolites, Antineoplastic; D003470:Culture Media; D011994:Recombinant Proteins; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase