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Our main interests are development of bioreactors for the production of therapeutic proteins and industrially useful bioresources using plant, insect and microorganism.
1. Production and glycoengineering of useful recombinant glycoproteins in plant cells, insect cells, microorganism and mammalian cells.
Recently, Human pharmaceutical proteins have been produced in lots of bioreactors (Mammalian cells, plants, insects, microorganisms, and so on). Almost all pharmaceutical proteins are glycosylated, and the glycan moiety contributes to the stability and bioactivity. Although plants, insects, and yeasts have advantages of the economical productivity as bioreactors comparing with mammalian cells, the recombinant pharmaceutical proteins actually have host-specific glycans, not human-type. The difference of the glycosylation pattern often causes reduction of the bioactivity and half-life in blood stream, and also shows immunogenicity in human. To solve these problems, we have to humanize such host-specific glycan structures. Our aim is to produce human oriented recombinant pharmaceutical proteins with basic glyco-science and glyco-engineering.
*click on the link to go to PubMed etc. if it is available.
Plant
(Arabidopsis thaliana, BY-2 cells, Nicotiana tabacum, Nicotiana benthamiana and MM2d cells)
1) Structural analysis of N- and O-linked oligosaccharides from several plant species
1.1) O-linked oligosaccharide analysis of rice alchol-extracted proteins
Kilcoyne, Shah, Gerlach, Bhavanandan, Nagaraj, Smith, Fujiyama, Sommer, Costello, Olszewski, Joshi. J. Plant Physiol. 166, 219-232, 2009
1.2) N-linked oligosaccharide analysis of Arabidopsis thaliana MM2-d cells
Fujiyama, Tanaka, Terashima, Seki. Plant Biotechnol. 24, 255-259, 2007
1.3) Analysis of sialylated oligosaccharides in Arabidopsis thaliana
Shah, Fujiyama, Flynn, Joshi. Nature Biotechol. 21, 1470-1, 2003
1.4) N-linked oligosaccharide analysis of tobacco BY2 cells
Misaki, Kimura, Fujiyama, Seki. Biosci. Biotechnol. Biochem. 65, 2482-2488, 2001
Palacpac, Kimura, Fujiyama, Yoshida, Seki. Biosci. Biotechnol. Biochem. 63, 35-39, 1999
2) Structural analysis of N-linked oligosaccharides from heterologously expressed pharmaceutical and industrially useful glycoproteins in plant cells.
2.1) HIV-1 vaccine (cholera toxin B subunit-membrane proximal (ectodomain) region of gp41 envelop protein, CTB-MPR649-684) in Nicotiana benthamiana
Matoba, Kajiura, Cherni, Doran, Bomsel, Fujiyama and Mor. Plant Biotechnol J. 7, 129-45, 2009
2.2) Edible vaccine against house dust mite (HDM) allergy (Der p 1 allergen protein) in rice
Yang, Kajiura, Suzuki, Hirose, Fujiyama and Takaiwa. Biochem Biophys Res Commun. 365, 334-9, 2008
2.3) Dengue vaccine (virus envelop protein, D2EIII) in Nicotiana benthamiana
Saejung, Fujiyama, Takasaki, Ito, Hori, Malasit, Watanabe, Kurane, Seki. Vaccine. 25, 6646-54, 2007
2.4) Poliovirus vaccine (poliovirus peptide, PVP) in Nicotiana tabacum
Fujiyama, Saejung, Yanagihara, Nakado, Misaki, Honda, Watanabe, Seki. J Biosci Bioeng. 101, 398-402, 2006
2.5) Mouse monoclonal antibody in BY2 cells
Fujiyama, Misaki, Katsura, Tanaka, Furukawa, Omasa, Seki. J Biosci Bioeng. 101, 212-8, 2006
2.6) Human lactoferrin in rice
Nandi, Yalda, Lu, Nikolov, Misaki, Fujiyama and Huang. Transfenic Res. 14, 237-49, 2005
Fujiyama, Sakai, Misaki, Yanagishita, Honda, Anzai and Seki. Biosci Biotechnol Biochem. 68, 2565-70, 2004
3) Glycoengineering of plant cells
3.1) Cloning and characterization of plant glycan modifying enzymes
Characterization of AtALG3
Kajiura, Seki, Fujiyama. Glycobiology. 20, 736-51, 2010
Characterization of NtGnTI
Dohi, Isoyama-Tanaka, Tokuda, Fujiyama. J Biosci Bioeng. 109, 388-91, 2010
Characterization of AtMANI
Kajiura, Koiwa, Nakazawa, Okazawa, Kobayashi, Seki, Fujiyama. Glycobiology. 20, 235-47, 2010
Analysis of CMP-sialic acid transporter-like proteins in rice
Takashima, Seino, Nakano, Fujiyama, Tsujimoto, Ishida, Hashimoto. Phytochemistry. 70, 1973-81, 2009
Cloning and Characterization AtCMPKDO-S
Misaki, Kajiura, Fujii, Fujiyama, Seki. J Biosci Bioeng. 108, 527-9, 2009
Cloning and gene expression analysis of tomato endo-beta-N-acetylglucosaminidase
Nakamura, Inoue, Maeda, Nakano, Hosoi, Fujiyama, Kimura. Biosci Biotechnol Biochem.73, 461-64, 2009
Characterization of Almond alfa-mannosidase
Misaki, Fujiyama, Yokoyama, Ido, Miyauchi, Yoshida, Seki. J Biosci Bioeng. 96, 187-92, 2003
Identification of Arabidopsis thaliana alfa-mannosidase
Fujiyama, Kira, Iizuka, Kimura, Seki. J Biosci Bioeng. 92, 401-4, 2001
3.2) Introduction of mammalian glycan modifying enzyme
Expression of human UDP-galactose transporter (hUGT1) in Nicotiana tabacum
Khalil, Kajiura, Fujiyama, Koike, Ishida, Tanaka. J Biosci Bioeng. 109, 159-69, 2010
Mouse antibody production in BY2 cells expressing human β1,4-galactosyltransferase
Fujiyama, Furukawa, Katsura, Misaki, Omasa, Seki. Biochem Biophys Res Commun. 358, 85-91, 2007
Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in BY2 cells
Misaki, Fujiyama, Seki. Biochem Biophys Res Commun. 339, 1184-9, 2006
Glycan analysis of suspension medium from human β1,4-galactosyltransferase expressing BY2 cells
Misaki, Kimura, Palacpac, Yoshida, Fujiyama, Seki. Glycobiology. 13, 199-205, 2003
Stable expression of human β1,4-galactosyltransferase in BY2 cells
Palacpac, Yoshida, Sakai, Kimura, Fujiyama, Yoshida, Seki. Proc Natl Acad Sci USA. 96, 4692-7, 1999
3.3) Functional glycobiology in plant
Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus
Kang, Frank, Kang, Kajiura, Vikram, Ueda, Kim, Bahk, Triplett, Fujiyama, Lee, Schaewen, Koiwa. Proc Natl Acad Sci USA. 105, 5933-8, 2008
Salt-adaptaion induces the glycan structures of N-glycans
Kimura, Watanabe, Kimura, Maeda, Murata, Fujiyama. Biosci Biotechnol Biochem. 72, 514-22, 2008
4) Engineering of the cellular localization of glycan modifying enzymes
ER localization of heterologously expressed mouse antibody in BY-2 cells
Fujiyama, Misaki, Sakai, Omasa, Seki. J Biosci Bioeng. 107, 165-72
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Insect cells
(Sf9 cells and Bombix mori)
1) Structural analysis of N-linked oligosaccharides from insect cells
N-linked glycan structures of mouse interferon-β produced in Bobbyx mori larvae
Misaki, Nagaya, Fujiyama, Yahagihara, Honda, Seki. Biochem Biophys Res Commun. 311, 979-86, 2003
2) Cloning and characterization of insect glycan modifying enzymes
Cloning and characterization of BmFDL
Nomura, Ikeda, Ishiyama, Mita, Tamura, Okada, Fujiyama, Usami. J Biosci Bioeng. 110, 386-91, 2010
Cloning and characterization of BmGlcNAcase1, 2
Okada, Ishiyama, Sezutsu, Usami, Tamura, Mita, Fujiyama, Seki. Biosci Biotechnol Biochem. 71, 1626-35, 2007
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Microorganisms
(Schizosaccharomyces pombe, Pichia species and E. coli)
1) Introduction of eukaryotic glycan modifying enzymes into E. coli and yeast
2) Efficient production of proteins from human, plant and insect cells
Cloning and characterization of BmGlcNAcase1, 2
Okada, Ishiyama, Sezutsu, Usami, Tamura, Mita, Fujiyama, Seki. Biosci Biotechnol Biochem. 71, 1626-35, 2007
2.1) Honey bee β1,3-GalT production in E. coli
Kimura, Sakamura, Ushijima, Hama, Kajiura, Fujiyama, Okihara, Hashimoto, Sugimoto, Yamada. Biosci Biotechnol Biochem. 71, 1111-4, 2007
2.2) Dengue vaccine (virus envelop protein, D2EIII) production in E. coli
Saejung, Puttikhunt, Prommool, Sojikul, Tanaka, Fujiyama, Malasit, Seki. J Biosci Bioeng. 102, 333-9, 2006
2.3) Human GnT-1 production in E. coli
Fujiyama, Ido, Misaki, Moran, Yanagihara, Honda, Nishimura, Yoshida, Seki. J Biosci Bioeng. 92, 569-74, 2001
2.4) Human ER Mannosidase production in E. coli
Fujiyama, Sakuradani, Moran, Yoshida, Seki. J Biosci Bioeng. 91, 419-21, 2001
Moran, Sakuradani, Jun, Fujiyama, Seki, Yoshida. 86, 277-83, 1998
2.5) Human β-GalT1 production in E. coli
Shibatani, Fujiyama, Nishiguchi, Seki, Maekawa. J Biosci Bioeng. 91, 85-71, 2001
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Mammalian cells
(CHO;Chinese Hamster Ovary) cells, COS7 (African Green Monkey SV40-transf’d kidney fibroblast cell line) cells, HEK (Human embryonic kidney) 293 cells and RAW (Mouse leukaemic monocyte macrophage cell line) cells)
1) Structural analysis of N-linked oligosaccharides of the recombinant proteins produced in mammalian cells
Kim, Tokunaga, Ozaki, Ishibashi, Honda, Kajiura, Fujiyama, Asano, Kumagai, Omasa, Ohtake. Appl. Microbiol. Biotechnol. 85, 535-42, 2010
2) Development of drug delivery system using plant-produced biopharmaceuticals
3) Functional analysis of recycling endosome
Misaki, Morimatsu, Uemura, Waguri, Miyoshi, Taniguchi, Matsuda, Taguchi. J. Cell. Biol. 191, 23-9, 2010
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2. Exploration of novel effective microorganisms for production of useful bioresources.
1) Yeast species isolated from mangrove in Thailand
Isolation of new yeast species
Kaewwichian, Yongmanitchai, Srisuk, Fujiyama, Limtong. FEMS Yeast Res. 10, 214-20, 2010
Limtong, Yongmanitchai, Kawasaki, Fujiyama. FEMS Yeast Res. 9, 504-10, 2009
One of the strains isolated in Thailand shows highly lipid production like linoleic acid (C17H31COOH, 18:2 (n-6)) and stearic acid (C17H35COOH).
2) Root nodule bacteria isolated from Iriomote island
We explore the possibilities for industrial application.
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