Growth requirement for methionine in human melanoma-derived cell lines with different levels of MMACHC expression and methylation.

Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.

SNHG3 could promote prostate cancer progression through reducing methionine dependence of PCa cells.

In recent years, morbidity and mortality of prostate cancer (PCa) have increased dramatically, while mechanistic understanding of its onset and progression remains unmet. LncRNA SNHG3 has been proved to stimulate malignant progression of multiple cancers, whereas its functional mechanism in PCa needs to be deciphered. In this study, our analysis in the TCGA database revealed high SNHG3 expression in PCa tissue. Further analysis in starBase, TargetScan, and mirDIP databases identified the SNHG3/miR-152-3p/SLC7A11 regulatory axis. FISH was conducted to assess the distribution of SNHG3 in PCa tissue. Dual-luciferase reporter gene and RIP assays confirmed the relationship among the three objects. Next, qRT-PCR and western blot were conducted to measure expression levels of SNHG3, miR-152-3p, and SLC7A11. CCK-8, colony formation, Transwell, and flow cytometry were carried out to assess proliferation, migration, invasion, methionine dependence, apoptosis, and the cell cycle. It was noted that SNHG3 as a molecular sponge of miR-152-3p stimulated proliferation, migration, and invasion, restrained methionine dependence and apoptosis, and affected the cell cycle of PCa cells via targeting SLC7A11. Additionally, we constructed xenograft tumor models in nude mice and confirmed that knockdown of SNHG3 could restrain PCa tumor growth and elevate methionine dependence in vivo. In conclusion, our investigation improved understanding of the molecular mechanism of SNHG3 modulating PCa progression, thereby generating novel insights into clinical therapy for PCa.

Histone methylation status of H3K4me3 and H3K9me3 under methionine restriction is unstable in methionine-addicted cancer cells, but stable in normal cells.

Methionine addiction is a fundamental and general hallmark of cancer. Methionine addiction prevents cancer cells, but not normal cells from proliferation under methionine restriction (MR). Previous studies reported that MR altered the histone methylation levels in methionine-addicted cancer cells. However, no study has yet compared the status of histone methylation status, under MR, between cancer cells and normal cells. In the present study, we compared the histone methylation status between cancer cells and normal fibroblasts of H3K4me3 and H3K9me3, using recombinant methioninase (rMETase) to effect MR. Human lung and colon cancer cell lines and human normal foreskin fibroblasts were cultured in control medium or medium with rMETase. The viability of foreskin fibroblasts was approximately 10 times more resistant to rMETase than the cancer cells in vitro. Proliferation only of the cancer cells ceased under MR. The histone methylation status of H3K4me3 and H3K9me3 under MR was evaluated by immunoblotting. The levels of the H3K4me3 and H3K9me3 were strongly decreased by MR in the cancer cells. In contrast, the levels of H3K4me3 and H3K9me3 were not altered by MR in normal fibroblasts. The present results suggest that histone methylation status of H3K4me3 and H3K9me3 under MR was unstable in cancer cells but stable in normal cells and the instability of histone methylation status under MR may determine the high methionine dependency of cancer cells to survive and proliferate.

Combination therapy of tumor-targeting Salmonella typhimurium A1-R and oral recombinant methioninase regresses a BRAF-V600E-negative melanoma.

Melanoma is a recalcitrant cancer. To improve and individualize treatment for this disease, we previously developed a patient-derived orthotopic xenograft (PDOX) model for melanoma. We previously reported the individual efficacy of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and recombinant methioninase (rMETase) for melanoma in the PDOX models of this disease. In the present study, we evaluated the efficacy of the combination of S. typhimurium A1-R with orally-administered rMETase (o-rMETase) for BRAF-V600E-negative melanoma in a PDOX model. Three weeks after implantation, 60 PDOX mouse models were randomized into six groups of 10 mice each: untreated control, temozolomide (TEM); o-rMETase; S. typhimurium A1-R; TEM + rMETase, S. typhimurium A1-R + rMETase. All treatments inhibited tumor growth compared to untreated control (TEM: p < 0.0001, rMETase: p < 0.0001, S. typhimurium A1-R: p < 0.0001, TEM + rMETase: p < 0.0001, S. typhimurium A1-R + rMETase: p < 0.0001). The most effective was the combination of S. typhimurium A1-R + o-rMETase which regressed this melanoma PDOX, thereby indicating a new paradigm for treatment of metastatic melanoma.

Extent and Instability of Trimethylation of Histone H3 Lysine Increases With Degree of Malignancy and Methionine Addiction.

BACKGROUND/AIM: Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect. Methionine addiction is due to excessive use of and dependence on methionine by cancer cells. In the present report, we correlated the extent of methionine addiction and degree of malignancy with the amount and stability of methylated histone H3 lysine marks. MATERIALS AND METHODS: We established low- and high-malignancy variants from a parental human pancreatic-cancer cell line and compared their sensitivity to methionine restriction and histone H3 lysine methylation status. RESULTS: A low-malignancy, low-methionine-addiction revertant of the parental pancreatic-cancer cell line had less methylated H3K9me3 and was less sensitive to methionine restriction effected by recombinant methioninase (rMETase) than the parental cell line. A high-malignancy variant of the pancreatic cancer cell line had increased methylated H3K9me3 and was more sensitive to methionine restriction by rMETase with regard to inhibition of proliferation and to instability of histone H3 lysine methylation than the parental cell line. Orthotopic malignancy in nude mice was reduced in the low-methionine-addiction revertant and greater in the high-malignancy variant than in the parental cell line. CONCLUSION: The present study indicates that the degree of malignancy is linked to the extent of methionine addiction and the level and instability of trimethylation of histone H3, suggesting these phenomena are linked as a fundamental basis of oncogenic transformation.

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players.

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.

Efficacy of Recombinant Methioninase (rMETase) on Recalcitrant Cancer Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models: A Review.

An excessive requirement for methionine (MET), termed MET dependence, appears to be a general metabolic defect in cancer and has been shown to be a very effective therapeutic target. MET restriction (MR) has inhibited the growth of all major cancer types by selectively arresting cancer cells in the late-S/G2 phase, when they also become highly sensitive to cytotoxic agents. Recombinant methioninase (rMETase) has been developed to effect MR. The present review describes the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) models of recalcitrant cancer, including the surprising result that rMETase administrated orally can be highly effective.

Tumor-Specific S/G2-Phase Cell Cycle Arrest of Cancer Cells by Methionine Restriction.

Cancer cells require elevated amounts of methionine (MET) and arrest their growth under conditions of MET restriction (MR). This phenomenon is termed MET dependence. Fluorescence-activated cell sorting (FACS) first indicated that the MET-dependent SV40-transformed cancer cells were arrested in the S and G2 phases of the cell cycle when under MR. This is in contrast to a G1-phase accumulation of cells, which occurs only in MET-supplemented medium at very high cell densities and which is similar to the G1 cell-cycle block which occurs in cultures of normal fibroblasts at high density. When the human PC-3 prostate carcinoma cell line was cultured in MET-free, homocysteine-containing (MET-HCY+) medium, there was an extreme increment in DNA content without cell division indicating that the cells were blocked in S phase. Recombinant methioninase (rMETase) treatment of cancer cells also selectively trapped cancer cells in S/G2: The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase trap, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S-phase-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil (5-FU) and which were highly effective in killing the cancer cells. In contrast, treatment of cancer cells with drugs in the presence of MET, only led to the majority of the cancer cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. MR has the potential for highly effective cell-cycle-based treatment strategy for cancer in the clinic.

High Efficacy of Recombinant Methioninase on Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models of Cancer.

Methionine (MET) is a general target in cancer due to the excess requirement of MET by cancer cells. MET has been effectively restricted by recombinant methioninase (rMETase) in mouse models of cell-line tumors. This chapter reviews the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) mouse models of human cancer. Ewing's sarcoma is a recalcitrant disease even though development of multimodal therapy has improved patients' outcome. A Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. rMETase effectively reduced tumor growth compared to the untreated control. The MET level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase treatment group. Body weight did not significantly differ at any time points between the two groups. A PDOX nude mouse model of a BRAF V600E-mutant melanoma was established in the chest wall of nude mice and also tested with rMETase in combination with a first-line melanoma drug, temozolomide (TEM). Combination therapy of TEM and rMETase was significantly more efficacious than either monotherapy. The results reviewed in this chapter demonstrate the clinical potential of rMETase.

Is the Hoffman Effect for Methionine Overuse Analogous to the Warburg Effect for Glucose Overuse in Cancer?

The general cancer-specific metabolic defect of methionine (MET) dependence is due to MET overuse for aberrant transmethylation reactions. The excess use of MET for aberrant transmethylation reactions apparently diverts methyl groups from DNA. The resulting global DNA hypomethylation is also a general phenomenon in cancer and leads to unstable genomes and aneuploid karyotypes. The excessive and aberrant use of MET in cancer is readily observed in [11C]-MET-PET imaging, where high uptake of [11C]-MET results in a very strong and selective tumor signal compared to normal tissue background for brain cancer and possibly other cancers. [11C]-MET is superior to [18C]-fluorodeoxyglucose (FDG) for PET imaging, suggesting that MET overuse in cancer ("Hoffman effect") is greater than glucose overuse in cancer ("Warburg effect").

Afterword: Oral Methioninase-Answer to Cancer and Fountain of Youth?

The elevated methionine (MET) requirement of cancer cells is termed MET dependence and is possibly the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo due to their elevated requirement for MET. rMETase can also potentiate chemotherapy drugs active in S phase due to the selective arrest of cancer cells in S/G2 phase during MET restriction (MR). We previously reported that rMETase, administrated by intraperitoneal injection (ip-rMETase), could inhibit tumor growth in mouse models of cancer including patient-derived orthotopic xenograft (PDOX) mouse models. We subsequently compared ip-rMETase and oral rMETase (o-rMETase) on a melanoma PDOX mouse model. o-rMETase was significantly more effective than ip-rMETase to inhibit tumor growth without overt toxicity. The combination of o-rMETase+ip-rMETase was significantly more effective than either monotherapy and completely arrested tumor growth. Thus, o-rMETase is effective as an anticancer agent with the potential of clinical development for chronic cancer therapy as well as for cancer prevention. o-rMETase may also have potential as an antiaging agent for healthy people, since MR has been shown to extend the life span of a variety of different organisms.

Altered Methionine Metabolism in Cancer Cells.

Many different types of cancer cells have been shown to be methionine (MET) dependent. Cancer cells, unlike normal cells, grow poorly or not at all when MET is restricted. Cancer cells have an elevated requirement for exogenous MET for growth, despite high levels of endogenous synthesis. This requirement reflects increased utilization of MET by cancer cells, analogous to increased utilization glucose by cancer cells (Warburg effect). To answer the critical question of whether MET-dependent cancer cells synthesize normal amounts of MET, we determined the levels of MET, S-adenosylmethionine (AdoMET), and S-adenosylhomocysteine (AdoHCY) that were synthesized by MET-dependent cancer cells under conditions of MET restriction. We demonstrated that MET-dependent cells synthesize a normal amount of endogenously synthesized MET but are still deficient in AdoMET. In contrast, exogenously supplied MET results in normal AdoMET levels. The ratio of AdoMET to AdoHCY is low in MET-dependent cells growing in MET-restricted medium but is normal when MET is supplied. Under conditions of MET restriction, the low AdoMET/AdoHCY ratio probably limits proliferation of MET-dependent cancer cells. The amount of free MET is also low in MET-dependent cancer cells under MET restriction. The elevated MET requirement for cancer cells may be due to enhanced overall rates of transmethylation compared to normal human cells. Thus, MET-dependent cancer cells have low levels of free MET, low levels of AdoMET, and elevated levels of AdoHCY under conditions of MET restriction probably due to overuse of MET for transmethylation reactions ("Hoffman effect"), thereby blocking cellular proliferation.

Linkage of Methionine Dependence and Other Features of Malignancy.

Cancer cells have an elevated methionine (MET) requirement compared to normal cells and are termed MET dependent. Cancer cells were isolated in MET-restricted (MR) medium that reverted from MET dependence to MET independence. Increased MET biosynthesis was not a prerequisite for reversion to MET independence, indicating that MET dependence was not due to reduced endogenous MET synthesis. MET-independent revertants of cancer cells concomitantly reverted for some of the other properties associated with malignancy: Of the 13 MET-independent revertants isolated 5 showed increased anchorage dependence as reflected by reduced cloning efficiencies in methylcellulose; 8 showed an increased serum requirement for optimal growth; 8 showed decreased cell density in medium containing high serum; and 3 altered their cell morphology significantly. Eight of the 13 revertants have increased chromosome numbers. Thus, by selecting for MET independence, it is possible to obtain heterogeneous reduced-malignancy revertants, indicating further a relationship between altered MET metabolism and other fundamental properties of oncogenic transformation.

Efficacy of Methionine-Restricted Diets on Cancers In Vivo.

Methionine (MET) dependence is the elevated requirement of cancer cells for MET. Cancer cells are arrested when MET is restricted in late S/G2 phase of the cell cycle. MET dependence may be the only known general metabolic defect in cancer. This chapter reviews the in vivo efficacy of dietary MET restriction (MR) to arrest human-cancer cell-line tumors and mouse tumors in nude mice. Human cancer xenografts in nude mice, when fed a MET-free diet, had greatly inhibited tumor growth. The body weight of mice on the MET-free diet was found to be maintainable by once-per-week administration of MET. These results suggested that MET dependence can be an important target for human cancer treatment. Yoshida sarcoma in nude mice on a MET-depleted diet regressed. MET depletion resulted in the extended survival of the tumor-bearing mice. These experiments are a prelude to further clinical studies of the efficacy of MR diets on cancer patients.

Dietary Methionine Restriction-Based Cancer Chemotherapy in Rodents.

The elevated methionine (MET) requirement for the growth of tumors, first observed by Sugimura in 1959, termed MET dependence, is a potentially highly effective therapeutic target. Proof of this principle is that when MET restriction (MR) was initially established in co-cultures of cancer and normal cells, MET dependence could be exploited to selectively kill cancer cells without killing co-cultured normal cells. MET-dependent cells become reversibly blocked in the late S/G2 phase of the cell cycle under MR enabling selective and effective S-phase chemotherapy against these blocked cancer cells. Subsequent MET repletion with an anti-mitotic drug was totally effective at selectively eliminating the MET-dependent cancer cells enabling the normal MET-dependent cells to take over the culture. We have also observed that the MET analog ethionine (ETH) is synergistic with MR in arresting the growth of the Yoshida sarcoma both in vitro and eliminating metastasis when transplanted to nude mice. MR increased the efficacy of cisplatinum (CDDP) against the MX-1 human breast carcinoma cell line when grown in nude mice. MR increased 5-fluorouracil (5-FU) efficacy on a human gastric cancer xenograft, SC-1-NU, in nude mice. MET-restricted total parenteral nutrition (MR TPN) was effective in Yoshida sarcoma-bearing rats. MR TPN with doxorubicin (DOX) and vincristine (VCR) resulted in significant tumor suppression and prolonged survival of Yoshida-sarcoma-bearing rats. These results were the basis of subsequent studies that used methioninase to effect MR for effective cancer therapy.

Total Methionine Restriction Treatment of Cancer.

This chapter reviews how total methionine (MET) restriction (MR) of a human brain tumor xenograft, effected by the combination of recombinant L-methionine-α-deamino-γ-lyase (rMETase) and a MET-free diet, greatly potentiates standard chemotherapy for brain tumors in mouse models. The growth of human brain tumor Daoy, SWB77, and D-54 xenografts in nude mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of an MR diet and rMETase and homocysteine to rescue normal cells and tissues. MET was depleted to below 5 µm by this treatment. MR for 10-12 days inhibited tumor growth, but did not prevent tumor regrowth after treatment cessation. A single dose of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was ineffective alone, was administered at the end of the MR regimen, and caused a more than 80-day growth delay for Daoy and D-54 and a 20-day growth delay for SWB77. The total MR treatment regimens also increased the efficacy of temozolomide (TMZ) against the SWB77 xenograft when administered at the end of the MET regimen.

Methioninase Gene Therapy.

Recombinant methioninase (rMETase) derived from Pseudomonas putida targets the elevated methionine (MET) requirement of cancer cells (methionine dependence) and has shown efficacy against a variety of cancer types in mouse models. To enhance the efficacy of rMETase, we constructed the pLGFP-METSN retrovirus encoding the P. putida methioninase (METase) gene fused with the green fluorescent protein (GFP) gene. pLGFP-METSN or control vector pLGFPSN was introduced into the human lung cancer cell line H460. The retrovirus-mediated METase gene transfer decreased the intracellular MET level of the cancer cells and consequently enhanced the efficacy of treatment with the rMETase protein. The rMETase gene was introduced into an adenovirus. rAd-METase transduction of human OVACAR-8 ovarian cancer cells and human fibrosarcoma HT1080 cells in vitro and in vivo resulted in high levels of METase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The combination of rAd-METase and rMETase was synergistic to kill these cells. Normal fibroblasts, on the other hand, appeared relatively resistant to the METase gene in the presence of rMETase. Adenoviral METase-transduced cancer cells were used in combination with selenomethionine, releasing highly toxic methylselenol, which killed both the cancer cells containing the METase gene and bystanders. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and cancer-cell apoptosis. Adenoviral METase-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. AdMETase/SeMET therapy was effective against Bcl-2-overproducing A549 lung cancer cells, which were resistant to staurosporine-induced apoptosis, with a strong bystander effect. The combination of Ad-METase/SeMET and doxorubicin (DOX) delayed the growth of the H460 human lung cancer, growing subcutaneously in nude mice. These results demonstrate the potential of methionine restriction (MR) for cancer treatment.

L-[Methyl-11C] Methionine-Positron-Emission Tomography (MET-PET).

Methionine (MET) dependence is a cancer-specific metabolic abnormality that is due to MET overuse for aberrant transmethylation reactions. [11C]-MET is very useful for positron-emission tomography (PET) due to MET overuse in malignant tumors. Many benefits of MET-PET have been demonstrated. MET-PET can differentiate recurrent glioma and necrosis. [11C]-MET-PET can also predict prognosis in gliomas better than [18F]-FDG PET. [11C]-MET-PET is better than MRI for predicting survival in low-grade glioma (LGG). MET-PET has greater specificity for detecting residual tumor after surgery than MRI.

Methioninase Cell-Cycle Trap Cancer Chemotherapy.

Cancer cells are methionine (MET) dependent compared to normal cells as they have an elevated requirement for MET in order to proliferate. MET restriction selectively traps cancer cells in the S/G2 phase of the cell cycle. The cell cycle phase can be visualized by color coding with the fluorescence ubiquitination-based cell cycle indicator (FUCCI). Recombinant methioninase (rMETase) is an enzyme that effectively degrades MET. rMETase induces S/G2-phase blockage of cancer cells which is identified by the cancer cells' green fluorescence with FUCCI imaging. Cancer cells in G1/G0 are the majority of the cells in solid tumors and are resistant to the chemotherapy. Treatment of cancer cells with standard chemotherapy drugs only led to the majority of the cancer cell population being arrested in G0/G1 phase, identified by the cancer cells' red fluorescence in the FUCCI system. The G0/G1-phase cancer cells are chemo-resistant. Tumor targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) was used to decoy quiescent G0/G1 stomach cancer cells growing in nude mice to cycle, with subsequent rMETase treatment to selectively trap the decoyed cancer cells in S/G2 phase, which made them highly sensitive to chemotherapy. Subsequent cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy was then administered to kill the decoyed and trapped cancer cells, which completely prevented or regressed tumor growth. In a subsequent experiment, a patient-derived orthotopic xenograft (PDOX) model of recurrent CDDP-resistant metastatic osteosarcoma was eradicated by the combination of Salmonella typhimurium A1-R decoy, rMETase S/G2-phase cell cycle trap, and CDDP cell kill. Salmonella typhimurium A1-R and rMETase pre-treatment thereby overcame CDDP resistance. These results demonstrate the effectiveness of the new chemotherapy paradigm of "decoy, trap, and kill" chemotherapy.

Development of Recombinant Methioninase for Cancer Treatment.

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. 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 recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. 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. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.