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