Teruyuki Nagamune | The University of Tokyo (original) (raw)

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Papers by Teruyuki Nagamune

Research paper thumbnail of Three proliferating cell nuclear antigen homologues from Metallosphaera sedula form a head-to-tail heterotrimer

Scientific reports, 2016

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role in DNA metabol... more Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role in DNA metabolism. Genome sequence analysis has revealed that some crenarchaea possess three PCNA genes in their genome, but it has been reported that three PCNAs do not always form a unique heterotrimer composed of one of each molecule. The thermoacidophilic archaeon, Metallosphaera sedula, has three PCNA homologue genes. Here, we demonstrated that the three PCNA homologues, MsePCNA1, MsePCNA2 and MsePCNA3, exclusively form a heterotrimer in a stepwise fashion; MsePCNA1 and MsePCNA2 form a heterodimer, and then MsePCNA3 binds to the heterodimer. We determined that the dissociation constants between MsePCNA1 and MsePCNA2, and between MsePCNA3 and the MsePCNA1:MsePCNA2 heterodimer are 0.29 and 43 nM, respectively. Moreover, the MsePCNA1, MsePCNA2 and MsePCNA3 heterotrimer stimulated M. sedula DNA ligase 1 activity, suggesting that the heterotrimer works as a DNA sliding clamp in the organism. The stabl...

Research paper thumbnail of Growth Control of Mammalian Cells via a Human Artificial Chromosome Harboring a Chimeric Receptor

Human artificial chromosome (HAC) is a promising tool to deliver therapeutic genes up to megabase... more Human artificial chromosome (HAC) is a promising tool to deliver therapeutic genes up to megabase-range size without any disruption of host genome. In this study, we employed an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells. We previously constructed 21ΔqHAC vector, which is derived from human chromosome 21 and housed in Chinese hamster ovary (CHO) cells. Here, we constructed a HAC vector harboring a ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer. Consequently, the cells showed BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The antibody/receptor chimera in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.

Research paper thumbnail of A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity

Chemical Communications, Nov 14, 2008

A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptide... more A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptides and proteins with high photolytic efficiency, which have many potential applications in the study and engineering of biological systems.

Research paper thumbnail of Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble ?-Cyclodextrin?Acrylamide Copolymer Beads

Biotechnol Lett, 2004

Solid-phase refolding methods are advantageous since they facilitate both separation of solid add... more Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Research paper thumbnail of Crystallization of a photosensitive nitrile hydratase from sp. N-771

Research paper thumbnail of Method of feeding a substrate into tubular bioreactor

Research paper thumbnail of Modulating the Actions of NK Cell-Mediated Cytotoxicity Using Lipid-PEG (n) and Inhibitory Receptor-Specific Antagonistic Peptide Conjugates

Biotechnology Progress, 2005

Regulating the cell surface modulates the actions of the biological cell response, derives practi... more Regulating the cell surface modulates the actions of the biological cell response, derives practical applications, and is of scientific interest. On the basis of our previous study using dioleylphosphatidylethanolamine poly(ethylene glycol) with multiple units of ethyleneoxide (DOPE-PEG (n)), we demonstrated the potency of DOPE-PEG (80) as a cell surface modulator. We prepared conjugates of DOPE-PEG (80) and two antagonistic peptides (C1, SGGGCLFNLPWLCG; C26, SGGGCPFSFLPWCG), specifically designed for the inhibitory receptor of natural killer (NK) cells. We confirmed that NK cells exhibited cytotoxicity against DOPE-PEG (80)-peptides-incorporated target cells. We further investigated whether the DOPE-PEG (80)-peptides could affect the cytotoxicity of NK cells in a concentration-dependent manner. C1 peptide showed downregulation of cytotoxicity at higher concentration, whereas C26 peptide exhibited the saturated cytotoxicity of NK cells at the same concentration. These results suggest that DOPE-PEG (80) can achieve the role of a cell surface modulator without inhibiting the action of conjugated molecules, despite their relatively small size.

Research paper thumbnail of Immobilized culture of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified surface

Biotechniques, Nov 1, 2003

Research paper thumbnail of Method of Assaying Interaction Between Proteins

Research paper thumbnail of Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Biotechnol Lett, 2004

Solid-phase refolding methods are advantageous since they facilitate both separation of solid add... more Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Research paper thumbnail of Monolayer Formation of Cytochrome b562 on Gold Surfaces and Its Reconstitution Reaction, Studied by Surface Plasmon Resonance Spectroscopy

Bull Chem Soc Jpn, 2002

ABSTRACT

Research paper thumbnail of Monomolecular Layer of the Alkylated Cytochrome b_ Formed at the Air-Water Interface

Bulletin of the Chemical Society of Japan, Apr 15, 1999

ABSTRACT

Research paper thumbnail of Fluidized bed cell culture apparatus with a drain valve for the removal of support material

Research paper thumbnail of 327758 Protein Polymerization Technique Using Horseradish Peroxidase Catalyzed Tyorsine Coupling Reaction

ABSTRACT Cross-linking reaction of proteins offers an enhancement effect of a distinct function o... more ABSTRACT Cross-linking reaction of proteins offers an enhancement effect of a distinct function of proteins due to the multivalent effect. In addition to that, conjugation of different kinds of proteins creates hybrid biomaterials possessing multiple functions of the building block proteins and such protein conjugates often show great performance caused by the synergistic effect. In this context, we have focused on developing an alternative protein cross-linking technique that enables to generate protein conjugates composed of multiple numbers of proteins. The horseradish peroxidase (HRP) mediated tyrosyl radical coupling reaction was utilized to achieve such protein cross-linking reaction. HRP catalyzes the oxidation reaction of various phenolic compounds by decomposing hydrogen peroxide and generates phenoxy radicals. These radicals then react with each other to form polymers. One of the 20 natural amino acids, tyrosine (Tyr or Y), has phenolic moiety in its side chain. Therefore Tyr can be recognized by HRP and polymerize upon HRP catalysis as well. Our strategy involves the genetic engineering of target proteins to introduce a highly flexible peptide tag containing tyrosine residues (hereafter called Y-tag). We anticipated that HRP recognizes the tyrosine residues in the Y-tag due to steric hindrance and electrostatic interaction between the target proteins and HRP. As model proteins, we selected E.coli alkaline phosphatase and streptavidin. The wild-type of these proteins showed almost no reactivity against HRP treatment, while the Y-tagged proteins showed high reactivity and resulted highly cross-linked protein conjugates. By using this methodology, we have demonstrated protein homoconjugation and protein heteroconjugation and also we very recently reported the control of the Y-tag and HRP mediated proteins conjugation reaction by using electrostatic interactions. In this report, we would like to summarize the recent progress of Y-tag and HRP mediated protein cross-linking techniques.

Research paper thumbnail of Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

Journal of Bioscience and Bioengineering, 2010

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay... more We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.

Research paper thumbnail of Knowledge based diagnosis of inoculum properties and sterilization time in lactic acid fermentation

Biotechnol Technique, 1992

Visiting researcher from Helsinki University of Technology at RIKEN SUMMARY A knowledge based sys... more Visiting researcher from Helsinki University of Technology at RIKEN SUMMARY A knowledge based system has been shown to be a powerful tool for diagnosing microbial activities during a fermentation process. Knowledge about lactic acid fermentation was collected by an experimental study of Lactobacillus casei. The effects of the inoculum properties and sterilization time on the cultivation were expressed in a form of a fuzzy rule-based knowledge network. The system was able to detect abnormal inoculum or sterilization conditions which caused malfunctions in the cultivations.

Research paper thumbnail of Creation of a thermostable firefly luciferase with pH-insensitive luminescent colors

Photochemistry and Photobiology, Mar 1, 2003

Research paper thumbnail of Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells

Biotechniques, 1996

We have designed and constructed a novel chimeric protein that consisted of a single domain of pr... more We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Research paper thumbnail of A new automatic system to evaluate the density of viable microorganisms in liquids

Systematic and Applied Microbiology, Sep 30, 1985

ABSTRACT

Research paper thumbnail of AN-P34 The generation of oligodendrocytes from induced pluripotent stem cells(Section I Animal and Plant Cell Cultures)

Journal of Bioscience and Bioengineering, Nov 1, 2009

Research paper thumbnail of Three proliferating cell nuclear antigen homologues from Metallosphaera sedula form a head-to-tail heterotrimer

Scientific reports, 2016

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role in DNA metabol... more Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role in DNA metabolism. Genome sequence analysis has revealed that some crenarchaea possess three PCNA genes in their genome, but it has been reported that three PCNAs do not always form a unique heterotrimer composed of one of each molecule. The thermoacidophilic archaeon, Metallosphaera sedula, has three PCNA homologue genes. Here, we demonstrated that the three PCNA homologues, MsePCNA1, MsePCNA2 and MsePCNA3, exclusively form a heterotrimer in a stepwise fashion; MsePCNA1 and MsePCNA2 form a heterodimer, and then MsePCNA3 binds to the heterodimer. We determined that the dissociation constants between MsePCNA1 and MsePCNA2, and between MsePCNA3 and the MsePCNA1:MsePCNA2 heterodimer are 0.29 and 43 nM, respectively. Moreover, the MsePCNA1, MsePCNA2 and MsePCNA3 heterotrimer stimulated M. sedula DNA ligase 1 activity, suggesting that the heterotrimer works as a DNA sliding clamp in the organism. The stabl...

Research paper thumbnail of Growth Control of Mammalian Cells via a Human Artificial Chromosome Harboring a Chimeric Receptor

Human artificial chromosome (HAC) is a promising tool to deliver therapeutic genes up to megabase... more Human artificial chromosome (HAC) is a promising tool to deliver therapeutic genes up to megabase-range size without any disruption of host genome. In this study, we employed an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells. We previously constructed 21ΔqHAC vector, which is derived from human chromosome 21 and housed in Chinese hamster ovary (CHO) cells. Here, we constructed a HAC vector harboring a ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer. Consequently, the cells showed BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The antibody/receptor chimera in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.

Research paper thumbnail of A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity

Chemical Communications, Nov 14, 2008

A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptide... more A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptides and proteins with high photolytic efficiency, which have many potential applications in the study and engineering of biological systems.

Research paper thumbnail of Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble ?-Cyclodextrin?Acrylamide Copolymer Beads

Biotechnol Lett, 2004

Solid-phase refolding methods are advantageous since they facilitate both separation of solid add... more Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Research paper thumbnail of Crystallization of a photosensitive nitrile hydratase from sp. N-771

Research paper thumbnail of Method of feeding a substrate into tubular bioreactor

Research paper thumbnail of Modulating the Actions of NK Cell-Mediated Cytotoxicity Using Lipid-PEG (n) and Inhibitory Receptor-Specific Antagonistic Peptide Conjugates

Biotechnology Progress, 2005

Regulating the cell surface modulates the actions of the biological cell response, derives practi... more Regulating the cell surface modulates the actions of the biological cell response, derives practical applications, and is of scientific interest. On the basis of our previous study using dioleylphosphatidylethanolamine poly(ethylene glycol) with multiple units of ethyleneoxide (DOPE-PEG (n)), we demonstrated the potency of DOPE-PEG (80) as a cell surface modulator. We prepared conjugates of DOPE-PEG (80) and two antagonistic peptides (C1, SGGGCLFNLPWLCG; C26, SGGGCPFSFLPWCG), specifically designed for the inhibitory receptor of natural killer (NK) cells. We confirmed that NK cells exhibited cytotoxicity against DOPE-PEG (80)-peptides-incorporated target cells. We further investigated whether the DOPE-PEG (80)-peptides could affect the cytotoxicity of NK cells in a concentration-dependent manner. C1 peptide showed downregulation of cytotoxicity at higher concentration, whereas C26 peptide exhibited the saturated cytotoxicity of NK cells at the same concentration. These results suggest that DOPE-PEG (80) can achieve the role of a cell surface modulator without inhibiting the action of conjugated molecules, despite their relatively small size.

Research paper thumbnail of Immobilized culture of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified surface

Biotechniques, Nov 1, 2003

Research paper thumbnail of Method of Assaying Interaction Between Proteins

Research paper thumbnail of Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Biotechnol Lett, 2004

Solid-phase refolding methods are advantageous since they facilitate both separation of solid add... more Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Research paper thumbnail of Monolayer Formation of Cytochrome b562 on Gold Surfaces and Its Reconstitution Reaction, Studied by Surface Plasmon Resonance Spectroscopy

Bull Chem Soc Jpn, 2002

ABSTRACT

Research paper thumbnail of Monomolecular Layer of the Alkylated Cytochrome b_ Formed at the Air-Water Interface

Bulletin of the Chemical Society of Japan, Apr 15, 1999

ABSTRACT

Research paper thumbnail of Fluidized bed cell culture apparatus with a drain valve for the removal of support material

Research paper thumbnail of 327758 Protein Polymerization Technique Using Horseradish Peroxidase Catalyzed Tyorsine Coupling Reaction

ABSTRACT Cross-linking reaction of proteins offers an enhancement effect of a distinct function o... more ABSTRACT Cross-linking reaction of proteins offers an enhancement effect of a distinct function of proteins due to the multivalent effect. In addition to that, conjugation of different kinds of proteins creates hybrid biomaterials possessing multiple functions of the building block proteins and such protein conjugates often show great performance caused by the synergistic effect. In this context, we have focused on developing an alternative protein cross-linking technique that enables to generate protein conjugates composed of multiple numbers of proteins. The horseradish peroxidase (HRP) mediated tyrosyl radical coupling reaction was utilized to achieve such protein cross-linking reaction. HRP catalyzes the oxidation reaction of various phenolic compounds by decomposing hydrogen peroxide and generates phenoxy radicals. These radicals then react with each other to form polymers. One of the 20 natural amino acids, tyrosine (Tyr or Y), has phenolic moiety in its side chain. Therefore Tyr can be recognized by HRP and polymerize upon HRP catalysis as well. Our strategy involves the genetic engineering of target proteins to introduce a highly flexible peptide tag containing tyrosine residues (hereafter called Y-tag). We anticipated that HRP recognizes the tyrosine residues in the Y-tag due to steric hindrance and electrostatic interaction between the target proteins and HRP. As model proteins, we selected E.coli alkaline phosphatase and streptavidin. The wild-type of these proteins showed almost no reactivity against HRP treatment, while the Y-tagged proteins showed high reactivity and resulted highly cross-linked protein conjugates. By using this methodology, we have demonstrated protein homoconjugation and protein heteroconjugation and also we very recently reported the control of the Y-tag and HRP mediated proteins conjugation reaction by using electrostatic interactions. In this report, we would like to summarize the recent progress of Y-tag and HRP mediated protein cross-linking techniques.

Research paper thumbnail of Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

Journal of Bioscience and Bioengineering, 2010

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay... more We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.

Research paper thumbnail of Knowledge based diagnosis of inoculum properties and sterilization time in lactic acid fermentation

Biotechnol Technique, 1992

Visiting researcher from Helsinki University of Technology at RIKEN SUMMARY A knowledge based sys... more Visiting researcher from Helsinki University of Technology at RIKEN SUMMARY A knowledge based system has been shown to be a powerful tool for diagnosing microbial activities during a fermentation process. Knowledge about lactic acid fermentation was collected by an experimental study of Lactobacillus casei. The effects of the inoculum properties and sterilization time on the cultivation were expressed in a form of a fuzzy rule-based knowledge network. The system was able to detect abnormal inoculum or sterilization conditions which caused malfunctions in the cultivations.

Research paper thumbnail of Creation of a thermostable firefly luciferase with pH-insensitive luminescent colors

Photochemistry and Photobiology, Mar 1, 2003

Research paper thumbnail of Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells

Biotechniques, 1996

We have designed and constructed a novel chimeric protein that consisted of a single domain of pr... more We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Research paper thumbnail of A new automatic system to evaluate the density of viable microorganisms in liquids

Systematic and Applied Microbiology, Sep 30, 1985

ABSTRACT

Research paper thumbnail of AN-P34 The generation of oligodendrocytes from induced pluripotent stem cells(Section I Animal and Plant Cell Cultures)

Journal of Bioscience and Bioengineering, Nov 1, 2009