Development of Recombinant Methioninase for Cancer Treatment

Robert M. Hoffman, Yuying Tan, Shukuan Li, Qinghong Han, Shigeo Yagi, Tomoaki Takakura, Akio Takimoto, Kenji Inagaki, Daizou Kudou

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)107-131
Number of pages25
JournalMethods in molecular biology (Clifton, N.J.)
Volume1866
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Methionine
Neoplasms
Half-Life
Holoenzymes
Enzymes
Endotoxins
Sepharose
Therapeutics
Gel Chromatography
Anions
Chromatography
Plasmids
Glutarates
Serum
Inbred BALB C Mouse
Pseudomonas putida
Lyases
Pyridoxal Phosphate
Initiator Codon
Freeze Drying

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Hoffman, R. M., Tan, Y., Li, S., Han, Q., Yagi, S., Takakura, T., ... Kudou, D. (2019). Development of Recombinant Methioninase for Cancer Treatment. Methods in molecular biology (Clifton, N.J.), 1866, 107-131. https://doi.org/10.1007/978-1-4939-8796-2_10

Development of Recombinant Methioninase for Cancer Treatment. / Hoffman, Robert M.; Tan, Yuying; Li, Shukuan; Han, Qinghong; Yagi, Shigeo; Takakura, Tomoaki; Takimoto, Akio; Inagaki, Kenji; Kudou, Daizou.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1866, 01.01.2019, p. 107-131.

Research output: Contribution to journalArticle

Hoffman, RM, Tan, Y, Li, S, Han, Q, Yagi, S, Takakura, T, Takimoto, A, Inagaki, K & Kudou, D 2019, 'Development of Recombinant Methioninase for Cancer Treatment', Methods in molecular biology (Clifton, N.J.), vol. 1866, pp. 107-131. https://doi.org/10.1007/978-1-4939-8796-2_10
Hoffman, Robert M. ; Tan, Yuying ; Li, Shukuan ; Han, Qinghong ; Yagi, Shigeo ; Takakura, Tomoaki ; Takimoto, Akio ; Inagaki, Kenji ; Kudou, Daizou. / Development of Recombinant Methioninase for Cancer Treatment. In: Methods in molecular biology (Clifton, N.J.). 2019 ; Vol. 1866. pp. 107-131.
@article{488b08aecd3a4910be64e4a827cfa280,
title = "Development of Recombinant Methioninase for Cancer Treatment",
abstract = "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.",
keywords = "3-Dimensional structure, Anticancer efficacy, Cancer, Crystallization, Industrial purification, L-Methionine α-deamino, MEGC-PEG, Methioninase, Methionine dependence, Methionine restriction, Methoxypolyethylene glycol, PEG, Polyethylene glycol, Pyridoxal-L-phosphate, Recombinant methioninase, rMETase, Succinimide glutarate, α-mercaptomethane lyase [EC4.4.1.11]",
author = "Hoffman, {Robert M.} and Yuying Tan and Shukuan Li and Qinghong Han and Shigeo Yagi and Tomoaki Takakura and Akio Takimoto and Kenji Inagaki and Daizou Kudou",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-8796-2_10",
language = "English",
volume = "1866",
pages = "107--131",
journal = "Methods in Molecular Biology",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Development of Recombinant Methioninase for Cancer Treatment

AU - Hoffman, Robert M.

AU - Tan, Yuying

AU - Li, Shukuan

AU - Han, Qinghong

AU - Yagi, Shigeo

AU - Takakura, Tomoaki

AU - Takimoto, Akio

AU - Inagaki, Kenji

AU - Kudou, Daizou

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - 3-Dimensional structure

KW - Anticancer efficacy

KW - Cancer

KW - Crystallization

KW - Industrial purification

KW - L-Methionine α-deamino

KW - MEGC-PEG

KW - Methioninase

KW - Methionine dependence

KW - Methionine restriction

KW - Methoxypolyethylene glycol

KW - PEG

KW - Polyethylene glycol

KW - Pyridoxal-L-phosphate

KW - Recombinant methioninase

KW - rMETase

KW - Succinimide glutarate

KW - α-mercaptomethane lyase [EC4.4.1.11]

UR - http://www.scopus.com/inward/record.url?scp=85061118184&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85061118184&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-8796-2_10

DO - 10.1007/978-1-4939-8796-2_10

M3 - Article

VL - 1866

SP - 107

EP - 131

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -