Development of Recombinant Methioninase for Cancer Treatment

  • Robert M. HoffmanEmail author
  • Yuying Tan
  • Shukuan Li
  • Qinghong Han
  • Shigeo Yagi
  • Tomoaki Takakura
  • Akio Takimoto
  • Kenji Inagaki
  • Daizou Kudou
Part of the Methods in Molecular Biology book series (MIMB, volume 1866)


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 (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.

Key words

Cancer Methionine dependence l-Methionine α-deamino α-mercaptomethane lyase [EC4.4.1.11] Methioninase Recombinant methioninase rMETase Polyethylene glycol PEG Methoxypolyethylene glycol Succinimide glutarate MEGC-PEG Pyridoxal-l-phosphate Methionine restriction Anticancer efficacy Industrial purification Crystallization 3-Dimensional structure 


  1. 1.
    Kreis W, Hession C (1973) Isolation and purification of L-methionine-a-deamino-g mercaptomethane-lyase (L-methioninase) from Clostridium. Cancer Res 33:1862–1865Google Scholar
  2. 2.
    Tan Y, Xu M, Tan X-Z, Tan X-Y, Wang X, Saikawa Y, Nagahama T, Sun X, Lenz M, Hoffman RM (1997) Overexpression and large-scale production of recombinant L-methionine-α-deamino-γ-mercaptomethane-lyase for novel anticancer therapy Protein Expr Purif 9:233–245Google Scholar
  3. 3.
    Ito S, Nakamura T, Eguchi Y (1976) Purification and characterization of methoninase from P. putida. J Biochem 79:1263–1272CrossRefGoogle Scholar
  4. 4.
    Nakamura T, Esaki N, Tanaka H, Soda K (1988) Specific labeling of the essential cysteine residue of L-methionine γ-lyase with a cofactor analogue, N-(bromo-acetyl) pyridoxamine phosphate. Biochemistry 27:1587–1591CrossRefGoogle Scholar
  5. 5.
    Tanaka H, Esaki N, Soda K (1977) Properties of L-methionine γ-lyase from Pseudomonas ovalis. Biochemistry 16:100–106CrossRefGoogle Scholar
  6. 6.
    Nakamura T, Esaki N, Sugiem K, Beresov T, Tanaka H, Soda K (1984) Purification of bacterial L-methionine-lyase. Anal Biochem 138:421–424CrossRefGoogle Scholar
  7. 7.
    Inoue H, Inagaki K, Sugimoto M, Esaki N, Soda K, Tanaka H (1995) Structural analysis of the L-methionine γ lyase gene from Pseudomonas putida. J Biochem 117:1120–1125CrossRefGoogle Scholar
  8. 8.
    Hori H, Takabayashi K, Orvis L, Carson DA, Nobori T (1996) Gene cloning and characterization of Pseudomonas put ida L-methionine-a-deamino-g-mercaptomethane-lyase. Cancer Res 56:2116–2122PubMedGoogle Scholar
  9. 9.
    Takakura T, Ito T, Yagi S, Notsu Y, Itakura T, Nakamura T, Inagaki K, Esaki N, Hoffman RM, Takimoto A (2006) High-level expression and bulk crystallization of recombinant l-methionine γ-lyase, an anticancer agent. Appl Microbiol Biotechnol 70:183–192CrossRefGoogle Scholar
  10. 10.
    Tan Y, Sun X, Xu M, An Z, Tan XZ, Tan XY, Han Q, Miljkovic DA, Yang M, Hoffman RM (1998) Polyethylene glycol conjugation of recombinant methioninase for cancer therapy. Protein Expr Purif 12:45–52CrossRefGoogle Scholar
  11. 11.
    Li S-K, Yang Z, Sun X, Tan Y, Yagi S, Hoffman RM (2004) Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation. Anal Biochem 330:264–272CrossRefGoogle Scholar
  12. 12.
    Yang Z, Sun X, Li S, Tan Y, Wang X, Zhang N, Yagi S, Takakura T, Kobayashi Y, Takimoto A, Yoshioka T, Suginaka A, Frenkel EP, Hoffman RM (2004) Circulating half-life of PEGylated recombinant methioninase holoenzyme is highly dose dependent on cofactor pyridoxal-5′-phosphate. Cancer Res 64:5775–5778CrossRefGoogle Scholar
  13. 13.
    Weiner MP, Anderson C, Jerpseth B, Wells S, Johnson Browne B, Vaillancourt P (1994) Studier pET system vectors and hosts. Strateg Mol Biol 7:41–43Google Scholar
  14. 14.
    Bovara R, Carrea G, Gioacchini AM, Riva S, Secundo F (1997) Activity, stability and conformation of methoxypoly (ethylene glycol)-subtilisin at different concentrations of water in dioxane. Biotechnol Bioeng 54:50–57CrossRefGoogle Scholar
  15. 15.
    Brumeanu T-D, Zaghouani H, Bona C (1995) Purification of antigenized immunoglobulins derivatized with mono methoxypolyethylene glycol. J Chromatogr A 696:219–225CrossRefGoogle Scholar
  16. 16.
    Sun X, Yang Z, Li S, Tan Y, Zhang N, Wang X, Yagi S, Yoshioka T, Takimoto A, Mitsushima K, Suginaka A, Frenkel EP, Hoffman RM (2003) In vivo efficacy of recombinant methioninase is enhanced by the combination of polyethylene glycol conjugation and pyridoxal 5′ phosphate supplementation. Cancer Res 63:8377–8383PubMedGoogle Scholar
  17. 17.
    Kudou D, Misaki S, Yamashita M, Tamura T, Takakura T, Yoshioka T, Yagi S, Hoffman RM, Takimoto A, Esaki N, Inagaki K (2007) Structure of the antitumour enzyme l-methionine γ-lyase from Pseudomonas putida at 1.8Å resolution. J Biochem 141: 535–544CrossRefGoogle Scholar
  18. 18.
    Watanabe N, Kamei S, Ohkubo A, Yamanaka M, Ohsawa S, Makino K, Tokuda K (1986) Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer. Clin Chem 32:1551–1554PubMedGoogle Scholar
  19. 19.
    Hoffman RM (2015) Development of recombinant methioninase to target the general cancer-specific metabolic defect of methioninase dependence: a 40-year odyssey. Expert Opin Biol Ther 15:21–31CrossRefGoogle Scholar
  20. 20.
    Hou KC, Zaniewski (1991) Endotoxin removal for anion exchange polymeric matrix. Biotechnol Appl Biochem 12:315–324Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Robert M. Hoffman
    • 1
    • 2
    Email author
  • Yuying Tan
    • 1
  • Shukuan Li
    • 1
  • Qinghong Han
    • 1
  • Shigeo Yagi
    • 1
  • Tomoaki Takakura
    • 3
  • Akio Takimoto
    • 3
  • Kenji Inagaki
    • 4
  • Daizou Kudou
    • 4
  1. 1.AntiCancer, Inc.San DiegoUSA
  2. 2.Department of SurgeryUniversity of CaliforniaSan DiegoUSA
  3. 3.Discovery Research LaboratoriesShionogi & Co., Ltd.HyogoJapan
  4. 4.Department of Biofunctional ChemistryGraduate School of Natural Science and Technology, Okayama UniversityOkayamaJapan

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