由金針菇(Flammulina Velutipes)表現的免疫調節功能蛋白(Fungal immunomodulatory protein,FIP,)可以使正常人類週邊血液的淋巴球(human peripheral blood lymphocytes) 及對小鼠脾臟細胞誘發干擾素(interferon-γ)增生，此外可以抑制Th2細胞所產生的細胞激素，但金針菇生長週期長且欲從金針菇中分離純化得到高純度之FIP-fve效率不高，每克金針菇可以純化出8.75微克 FIP，因此本研究利用與金針菇同屬真菌之嗜甲基酵母菌為宿主來表現生產FIP，將FIP-fve基因建構於含AOXI啟動子 P.pastoris pPIC9K 質體中，此質體經轉形進入P.pastoris GS115，經篩選確定染色體中含有FIP-fve基因之菌株命名為P. pastoris-AαFIPfve。此菌株用搖瓶培養在30℃、300rpm，培養5天後可得到乾菌重12.83(g/l)，經金屬螯合層析純化後的蛋白質有1.615(mg/g cell)。

Fungal immunomodulatory protein (FIP-fve) can be isolated and purified from Flammulina velutipes with very low yield (8.75μl/g biomass) . FIP-fve contains 114 amino acid residus with an acetylated C-terminal . FIP-fve can stimulate the proliferation of human peripheral blood lymphocytes , induce mice spleen cells to express interferon-γ , and inhibit Th2 cell from producing cytokine like IL-4, IL-5,IL-6,IL-10 and IL-13 . In this thesis methylotrophic yeast Pichia pastoris was employed to express FIP-fve. An integrative vector containing alcohol oxidase (AOX1) promoter controlled FIP-fve gene was contracted and transformed into P. pastoris GS115. Since α-factor prepro signal peptides of the Saccharomyces cerevisiae was employed to direct the secretion of FIP-fve, FIP-fve was detected in the fermentation broth after 1%(v/v) methanol induction. One gram of P. pastoris can produce 1~2 mg FIP-fve after purified by immobilized metal ion affinity chromatography (IMAC). FIP-fve were observed three major bands in SDS-PAGE including 14kDa, 18kDa and 24kDa . In western bolting analysis using either anti-His antibody or anti-FIPfve antibody as probe also detected these three protein bands

Cereghino J. L., Merryweather J. P., Cooit D. J., Heeberlein U. A., Masiaarz F. R., Mullenbath G. T., Urdea M. S., Valezuela P., and Barr P. J. 1984.α-factor-dirceted synthesis and secretion of mature foreign protein in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. 81:4642-4646

Cereghino J. L., Cregg J. M.2000.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev 24:45-66

Charlwood J., Bryant D., Skehel J. M., Camilleri P.2001.Analysis of N-linked oligosaccharides : progress towards the characterization of glycoprotein-linked carbohydrates.Biomol Eng 18:229-240

Cregg J. M, Madden K. R. 1989. Use of site-specific recombination to regenerate selectable markers. Mol Gen Genet 219:320-3

Cregg J. M., Vedvick T. S., Raschke W. C.1993. Recent advances in the expression of foreign gene in Pichia pastois. Bio/Technology 11:905-910

Boehm T, Pirie-Shepherd S, Trinh LB, Shiloach J, Folkman J.1999. Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast 15:563-72

Bradford M.M., A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976

Hsu H. C., Hsu C. I., Lin R. H., Kao C. L., and Lin J. Y.1997. Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem. J.323:557-565

Ikekawe T., Nakanishi M., Uehara U., Chihara G., Fukuoka F.1968. Antitumor action of some basidiomycetes, especially Phllinus inteus. Japanese Journal of Cancer Research 59(2):155-157

Fujimiya Y., Suzuki Y., Oshiman K., Kobori H., Moriguchi K., Nakashima H., Matumoto Y. Takahara S., Ebina T., Katakura R. 1998. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natura killer cell activation and apoptosis.Cancer Immunol Immunother 46:147-159.

Kohsuke Kino, Akio Yamashita, Kyoko Yamaoka, Junko Watanabe, Shizuko Tanaka, KerKryO,Kimiko Shimizu, and Hajime Tsunoo.1989. Isolation and Characterization of a New Immunomodulatory Protein, Ling Zhi-8 (LZ-8), from Ganoderrna Zucidiurn.The Journal of Biological chemistry 264: 472-478.

Lin W. H., Hung C. H., Hsu C. I. and Lin J. Y.1997. Dimerization of the N-terminal Amphipathic a-Helix Domain of the Fungal Immunomodulatory Protein from Ganoderma tsugae (Fip-gts) Defined by a Yeast Two-hybrid System and Site-directed Mutagenesis. The Journal of Biological chemistry.272:20044-20048.

Lemontt J. F., Wei C. M., Lawman M. J., Gray P. W.1985.Expression of active human uterine tissue plaminogen activator in yeast. DNA.4:419-428.

Ko J. L., Hsu C. I., Lin R. H., Kao C. L., and Lin J. Y.1995. A new fungal immunomodulatory protein, FIP-fve isolated from the edible
mushroom, Flammulina velutipes and its complete amino acid sequence . Eur. J. Biochem. 228: 244-249

Mitsuaki Kimura, Satoru Tsuruta and Takami Yoshida.1999.IL-4 production by PBMCS on stimulation with mite allengen is correlated with the level of serum IgE antibody against mite in children with bronchial asthma. J. Allergy Clin. Immun.150:327-32

Ostergaard S., Olsson L., Nielsen J.2000. Metabolic Engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 64:34-50

Shizuko Tanaka, Kerry KO, Kohsuke Kino, Kazuko Tsuchiya, Akio Yamashita, Akira Murasugi, Sadatoshi Sakuma, and Hajime Tsunoo.1989
Complete Amino Acid Sequence oafn Immunomodulatory Protein, Ling Zhi-8 (LZ-8) The journal of biological chemistry.264:16372-16377.

Simon Ostergaard, Lisbeth Olsson and Jens Nielsen.2000. Metabolic Engineering of Saccharomyces cerevisiae. Microbiology and molecular biology reviews.34-50.

Palasingam Paaventhan1, Jeremiah S. Joseph1, See Voon Seow, Shai Vaday, Howard Robinson, Kaw Yan Chua and Prasanna R. Kolatkar. 2003. A 1.7 A° Structure of Fve, a Member of the New Fungal Immunomodulatory Protein Family. J. Mol. Biol.332:461–470

Romanes M. A.1995.Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527-533.

Wang S. Y., Hsu M. L., Hsu H. C., Tzeng C. H., Lee S. S., Shiao M. S., Ho C. K.1997.The anti-tumor effect of Ganoderma lucidum is mediated by cytokines relased from activated macrophage and T lymphocytes.Internation Journal of Cancer 70:699-705

Van Arsdell, J N., S. Kwork, V.L. Schweichart, M. B. Ladner, D. H. Gelfand, and M. A. Innis. 1987. Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichderma reesei. Biotechnology 5:60-4

Wegner G.1990.Emerging applications of the methlotrophic yeasts. FEMS Microbiol Rev ; 186:37-44

Wang P. H., Hsu C. I., Tang S. C., Huang Y. L., Lin J.Y., And Ko J. L. 2004. Fungal Immunomodulatory Protein from Flammulina velutipes Induces Interferon-ç Production through p38 Mitogen-Activated Protein Kinase Signaling Pathway. J. Agric. Food Chem. 52: 2721-2725

林自威. S. cerevisiae與P. pastoris高密度發酵生產葡萄糖氧化酶及其酵素特性比較. 國立台灣科技大學碩士畢業論文;2004;6月

李承軒. 嗜甲基酵母菌表現融合透明顫菌血紅素之葡萄糖氧化酶(VHb-GOX)之研究. 國立台灣科技大學碩士畢業論文;2006;7月

吳誌明. 工程觀點探討嗜甲基酵母菌之蛋白質生產表現. 國立台灣科技大學博士畢業論文;2004;7月

林采蔆. 靈芝屬免疫調節蛋白GMI與GFP-1基因之選殖與Pichia pastoris之異源表現. 國立台灣大學碩士畢業論文;2004;7月