OVEREXPRESSION AND LARGE-SCALE PRODUCTION OF RECOMBINANT THIONINE-ALPHA-DEAMINO-GAMMA-MERCAPTOMETHANE-LYASE FOR NOVEL ANTICANCER THERAPY

The goal of the next generation of cancer chemotherapy is effective tu mor-selectivity. A tumor-selective target with high therapeutic potent ial is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine i n tumors methionine dependence. To selectively target the methionine d ependence of tumors for treatment on a large-scale preclinical and cli nical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyas e (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coil using the polymerase chain reaction (PCR). The MET ase gene was then ligated into the pT7-7 overexpression plasmid contai ning the T7 RNA polymerase promoter and recloned in E. coil strain BL2 1(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recomb inant methioninase (rMETase) and distinguished rMETase-overproducer fr om rMETase-negative colonies. A scale-up production protocol which con tained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiCl ean Etox endotoxin-affinity chromatography columns has been establishe d. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protoc ol can produce therapeutic rMETase at the multi-gram level per batch w ith high yield (> 60%), high purity (> 98%), high stability, and low e ndotoxin. Purified rMETase is stable to lyophilization. The t(1/2) of rMETase was 2 h when rMETase was administered by iv injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of h uman tumors tested including those from lung, colon, kidney, brain, pr ostate, and melanoma were sensitive to rMETase. In contrast normal cel ls were insensitive to rMETase in vitro and correspondingly, no toxici ty was detected in vivo at the effective doses. In conclusion, the ove rexpression clone and large-scale production protocols for rMETase hav e enabled rMETase to be used as a tumor-selective therapeutic with bro ad indication and high promise for effective, low-toxicity human cance r therapy. (C) 1997 Academic Press.