Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy.

摘要: The goal of the next generation cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential elevated methionine requirement tumor cells relative to normal cells. We have termed for in tumors dependence. To selectively dependence treatment on a large-scale preclinical and clinical basis, L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned Escherichia coli using polymerase chain reaction (PCR). METase was then ligated into pT7-7 overexpression plasmid containing T7 RNA promoter recloned E. strain BL21(DE3). pAC-1 clone isolated by its yellow-orange color which due enrichment pyridoxal phosphate-containing recombinant methioninase (rMETase) distinguished rMETase-overproducer rMETase-negative colonies. scale-up production protocol contained heat step, two DEAE Sepharose FF ion-exchange, one ActiClean Etox endotoxin-affinity chromatography columns established. produces rMETase at approximately 10% total soluble protein up 1 g/liter shake-flask culture. can produce multi-gram level per batch yield (> 60%), purity 98%), stability, low endotoxin. Purified stable lyophilization. t1/2 2 h when administered i.v. injection mice. Studies antitumor efficacy vitro vivo human xenografted nude mice demonstrated that all types tested including those lung, colon, kidney, brain, prostate, melanoma were sensitive rMETase. In contrast, insensitive correspondingly, no toxicity detected doses. conclusion, protocols enabled be used as broad indication promise effective, low-toxicity therapy.