茉莉酮酸是茉莉酸以及其衍生物的總稱。作為訊息調控因子，調控植物組織生長發育、抵禦食植動物的攝食並且調控因外界因子入侵所引發的系統性免疫反應。茉莉酸能夠與異白胺酸結合形JA-Ile並進入細胞核中，引發JAZ蛋白 (jasmonate ZIM-domain) 降解，使被JAZ蛋白抑制的轉譯因子活化，啟動下游基因表現。茉莉酸與其衍生物可由AtABCG16進行運送，將茉莉酸運出細胞外，調控細胞內茉莉酸的濃度。目前AtABCG16如何專一性辨識茉莉酸之運輸機制尚不清楚，其運輸方向性調控方式亦尚無相關報導，仍需更多結構與生化的研究來探討。本研究分別利用三種酵母菌蛋白表現系統Saccharomyces cerevisiae、Schizosaccharomyces pombe與Pichia pastoris，建立表現與純化AtABCG16重組蛋白的流程，期望能建立AtABCG16蛋白表現平台，以進行生化功能分析。在完成各酵母菌系統的質體建構與優化誘導條件後，已成功取得含AtABCG16-GFP的酵母菌膜樣本。經界面活性劑萃取膜蛋白後分析，在S. cerevisiae 與 P. pastoris表現系統表現之AtABCG16-GFP樣本，有較多蛋白降解的狀況，而S. pombe則產出較為完整的目標蛋白。以螢光分子篩管柱層析法分析該目標蛋白之分子量約200 kDa，顯示AtABCG16蛋白可能形成二聚體構型。利用金屬螯合層析法進行純化後，目前僅能取得少量目標單白，仍需針對現有製備流程進行優化，期望這些研究成果能被應用於往後其他真核生物膜蛋白的生化研究。

Jasmonates, which include jasmonic acid (JA) and its derivative, regulate the growth, development, defense, and immunity of plants. JA could conjugate with isoleucine (Ile) to form JA-Ile which could enter nuclear and cause the degradation of JAZ (jasmonate ZIM-domain protein). Degradation of JAZ would induce the expression of JA response genes and trigger the downstream signal transduction. JA was reported to be exported by AtABCG16 to regulate JA concentration in the cell. However, the molecular mechanisms of the JA transport and substrate specificity of AtABCG16 are still unclear. Structural and biochemical evidence is required to reveal the substrate selectivity and transport direction of AtABCG16. In this study, three common yeast recombinant expression hosts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris were used to express recombinant AtABCG16 heterologously. We aimed to set up the system for AtABCG16 expression. After establishing the expression constructs and induction protocols for each yeast system, we could successfully isolate the yeast membrane fractions containing AtABCG16-GFP from all three yeast expression systems. However, the AtABCG16 expressed by S. cerevisiae and P. pastoris formed heavy aggregation and degradation after detergent solubilization. Interestingly, the membrane proteins extracted from S. pombe membrane showed a major fluorescence distribution at about 200 kDa in size exclusion chromatography (SEC), indicating that AtABCG16 might form a dimer conformation. In addition, the solubilized AtABCG16-GFP were harvested by immobilized metal affinity chromatography. The results showed that small amounts of S. pombe-derived AtABCG16-GFP could be obtained. The optimization of protein production processes should be further studied. Taken together, this study established a procedure to obtain the recombinant ABCG transporters in yeast. These results could be utilized in the biochemical researches of eukaryotic transmembrane proteins.

陳昱安，以蛋白結構探討坎氏弧菌抗原HSP60之免疫原性，國立成功大學生物科技與產業科學系碩士論文，2019。

Abdala, G., Miersch, O., Correa, N. and Rosas, S. Detection of jasmonic acid in cultures of Escherichia coli and Saccharomyces cerevisiae. Natural Product Letters 14, 55-63, 1999.

Böer, E., Steinborn, G., Kunze, G. and Gellissen, G. Yeast expression platforms. Applied Microbiology and Biotechnology 77, 513-523, 2007.

Baghban, R., Farajnia, S., Rajabibazl, M., Ghasemi, Y., Mafi, A., Hoseinpoor, R., Rahbarnia, L. and Aria, M. Yeast Expression Systems: Overview and Recent Advances. Molecular Biotechnology 61, 365-384, 2019.

Baldock, D., Sheldon, J., Tailor, R., Green, K., Ray, J., Singh, S. and Brocklehurst, K. An improved method for the expression screening of membrane protein-GFP fusions in yeast. BioRxiv 10, 172114-172131, 2017.

Bill, R.M., Henderson, P.J., Iwata, S., Kunji, E.R., Michel, H., Neutze, R., Newstead, S., Poolman, B., Tate, C.G. and Vogel, H. Overcoming barriers to membrane protein structure determination. Nature Biotechnology 29, 335-340, 2011.

Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., MacAskill, A., Napier, J.A., Beale, M.H., Lilley, K.S. and Dupree, P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiology 137, 104-116, 2005.

Catty, P. and Goffeau, A. Identification and phylogenetic classification of eleven putative P-type calcium transport ATPase genes in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Bioscience Reports 16, 75-85, 1996.

Choi, B. K., Bobrowicz, P., Davidson, R.C., Hamilton, S.R., Kung, D.H., Li, H., Miele, R.G., Nett, J.H., Wildt, S. and Gerngross, T.U. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proceedings of the National Academy of Sciences of the United States of America 100, 5022-5027, 2003.

Cregg, J.M., Cereghino, J.L., Shi, J. and Higgins, D.R. Recombinant protein expression in Pichia pastoris. Molecular Biotechnology 16, 23-52, 2000.

Davidson, A.L. and Chen, J. ATP-binding cassette transporters in bacteria. Annual Review of Biochemistry 73, 241-268, 2004.

Drew, D., Lerch, M., Kunji, E., Slotboom, D.J. and de Gier, J.W. Optimization of membrane protein overexpression and purification using GFP fusions. Nature Methods 3, 303-313, 2006

Drew, D., Newstead, S., Sonoda, Y., Kim, H., Von Heijne, G. and Iwata, S. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nature Protocols 3, 784-798, 2008.

Duina, A.A., Miller, M.E. and Keeney, J.B. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48, 2014.

Ellinger, P., Kluth, M., Stindt, J., Smits, S.H. and Schmitt, L. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris. Public Library of Science One 8, e60620, 2013.

Freigassner, M., Pichler, H. and Glieder, A. Tuning microbial hosts for membrane protein production. Microbial Cell Factories 8, 69-91, 2009.

Frelet, A. and Klein, M. Insight in eukaryotic ABC transporter function by mutation analysis. Federation of European Biochemical Societies Letters 580, 1064-1084, 2006.

Geertsma, E.R., Groeneveld, M., Slotboom, D. J. and Poolman, B. Quality control of overexpressed membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 105, 5722-5727, 2008.

Gleeson, M.A., White, C.E., Meininger, D.P. and Komives, E.A. Generation of protease-deficient strains and their use in heterologous protein expression. Pichia Protocols, Springer, Germany, 81-94, 1998.

Gräfe, K. and Schmitt, L. The ABC transporter G subfamily in Arabidopsis thaliana. Journal of Experimental Botany 260, 1-14, 2020.

Gräfe, K., Shanmugarajah, K., Zobel, T., Weidtkamp-Peters, S., Kleinschrodt, D., Smits, S.H. and Schmitt, L. Cloning and expression of selected ABC transporters from the Arabidopsis thaliana ABCG family in Pichia pastoris. Public Library of Science One 14, e0211156, 2019.

Grossmann, G., Opekarova, M., Malinsky, J., Weig‐Meckl, I. and Tanner, W. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast. The European Molecular Biology Organization Journal 26, 1-8, 2007.

Hamilton, S.R., Cook, W.J., Gomathinayagam, S., Burnina, I., Bukowski, J., Hopkins, D., Schwartz, S., Du, M., Sharkey, N.J. and Bobrowicz, P. Production of sialylated O-linked glycans in Pichia pastoris. Glycobiology 23, 1192-1203, 2013.

Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H. and Choi, B. K. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441-1443, 2006.

Hartl, F.U. and Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-1858, 2002.

Higgins, C.F. ABC transporters: physiology, structure and mechanism–an overview. Research in Microbiology 152, 205-210, 2001.

Higgins, C.F., Hiles, I.D., Salmond, G.P., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D. and Bell, A.W. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature 323, 448-450, 1986.

Irani, Z.A., Kerkhoven, E.J., Shojaosadati, S.A. and Nielsen, J. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins. Biotechnology and Bioengineering 113, 961-969, 2016.

Ji, H., Peng, Y., Meckes, N., Allen, S., Stewart, C.N. and Traw, M.B. ATP-dependent binding cassette transporter G family member 16 increases plant tolerance to abscisic acid and assists in basal resistance against Pseudomonas syringae DC3000. Plant Physiology 166, 879-888, 2014.

Kadaba, N.S., Kaiser, J.T., Johnson, E., Lee, A. and Rees, D.C. The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation. Science 321, 250-253, 2008.

Kaishima, M., Ishii, J., Matsuno, T., Fukuda, N. and Kondo, A. Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity. Scientific Reports 6, 35932-35946, 2016.

Kawate, T. and Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673-681, 2006.

Kintzer, A.F. and Stroud, R.M. Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 258-262, 2016.

Klappe, K., Hummel, I., Hoekstra, D. and Kok, J.W. Lipid dependence of ABC transporter localization and function. Chemistry and Physics of Lipids 161, 57-64, 2009.

Lee, J.Y., Kinch, L.N., Borek, D.M., Wang, J., Wang, J., Urbatsch, I.L., Xie, X.S., Grishin, N.V., Cohen, J.C., Otwinowski, Z., Hobbs, H.H. and Rosenbaum, D.M. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 533, 561-564, 2016.

Li, C., Lin, Y., Zheng, X., Pang, N., Liao, X., Liu, X., Huang, Y. and Liang, S. Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris. BioMed Central Biotechnology 15, 88, 2015.

Li, Q., Zheng, J., Li, S., Huang, G., Skilling, S.J., Wang, L., Li, L., Li, M., Yuan, L. and Liu, P. Transporter-Mediated Nuclear Entry of Jasmonoyl-Isoleucine Is Essential for Jasmonate Signaling. Molecular Plant 10, 695-708, 2017.

Locher, K.P., Lee, A.T. and Rees, D.C. The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296, 1091-1098, 2002.

Marley, J., Lu, M. and Bracken, C. A method for efficient isotopic labeling of recombinant proteins. Journal of Biomolecular Nuclear Magnetic Resonance 20, 71-75, 2001.

Mongrand, S., Morel, J., Laroche, J., Claverol, S., Carde, J. P., Hartmann, M. A., Bonneu, M., Simon Plas, F., Lessire, R. and Bessoule, J. J. Lipid rafts in higher plant cells purification and characterization of triton X-100-insoluble microdomains from tobacco plasma membrane. Journal of Biological Chemistry 279, 36277-36286, 2004.

Moreno, S. Molecular genetc analysis of fission yeast Schizosaccharomyces pombe. Methods in Enzymology 194, 773-782, 1991.

Pandey, A., Shin, K., Patterson, R.E., Liu, X. Q. and Rainey, J.K. Current strategies for protein production and purification enabling membrane protein structural biology. Biochemistry and Cell Biology 94, 507-527, 2016.

Park, J., Lee, Y., Martinoia, E. and Geisler, M. Plant hormone transporters: what we know and what we would like to know. BioMed Central Biology 15, 93, 2017.

Parker, J.L. and Newstead, S. Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies. Protein Science 23, 1309-1314, 2014.
Petersen, J. and Russell, P. Growth and the Environment of Schizosaccharomyces pombe. Cold Spring Harbor Protocols 10, 210-226, 2016.

Piirainen, M.A., Boer, H., de Ruijter, J.C. and Frey, A.D. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae. Glycoconjugate Journal 33, 189-199, 2016.

Ramón, A. and Marín, M. Advances in the production of membrane proteins in Pichia pastoris. Biotechnology Journal 6, 700-706, 2011.

Rath, A., Glibowicka, M., Nadeau, V.G., Chen, G. and Deber, C.M. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 106, 1760-1765, 2009.

Rea, P.A. Plant ATP-binding cassette transporters. Annual Review of Plant Biology 58, 347-375, 2007.

Rosano, G.L. and Ceccarelli, E.A. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology 5, 172, 2014.

Ruan, J., Zhou, Y., Zhou, M., Yan, J., Khurshid, M., Weng, W., Cheng, J. and Zhang, K. Jasmonic Acid Signaling Pathway in Plants. International Journal of Molecular Sciences 20, 2479-2494, 2019.

Sahdev, S., Khattar, S.K. and Saini, K.S. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Molecular and Cellular Biochemistry 307, 249-264, 2008.

Saito, H., Oikawa, T., Hamamoto, S., Ishimaru, Y., Kanamori-Sato, M., Sasaki-Sekimoto, Y., Utsumi, T., Chen, J., Kanno, Y., Masuda, S., Kamiya, Y., Seo, M., Uozumi, N., Ueda, M. and Ohta, H. The jasmonate-responsive GTR1 transporter is required for gibberellin-mediated stamen development in Arabidopsis. Nature Communications 6, 6095-6176, 2015.
Sambrook, J., Fritsch, E.F. and Maniatis, T. Plasmids and their usefulness in molecular cloning. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 31-122, 1989a.

Sambrook, J., Fritsch, E.F. and Maniatis, T. In vitro amplification of DNA by the polymerase chain reaction. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 18-76, 1989b.

Sezonov, G., Joseleau-Petit, D. and d'Ari, R. Escherichia coli physiology in Luria-Bertani broth. Journal of Bacteriology 189, 8746-8749, 2007.

Sánchez-Fernández, R.o., Davies, T.E., Coleman, J.O. and Rea, P.A. The Arabidopsis thaliana ABC protein superfamily, a complete inventory. Journal of Biological Chemistry 276, 30231-30244, 2001.

Stewart, M.Q., Esposito, R.D., Gowani, J. and Goodman, J.M. Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments. Journal of Cell Science 114, 2863-2868, 2001.

Swinehart, D.F. The beer-lambert law. Journal of Chemical Education 39, 333, 1962.

Tanaka, Y., Iwaki, S. and Tsukazaki, T. Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein. Structure 25, 1455-1460, 2017.

Tang, H., Wang, S., Wang, J., Song, M., Xu, M., Zhang, M., Shen, Y., Hou, J. and Bao, X. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae. Scientific Reports 6, 25654-25666, 2016.

Thomas, J.A. and Tate, C.G. Quality control in eukaryotic membrane protein overproduction. Journal of Molecular Biology 426, 4139-4154, 2014.

Titapiwatanakun, B. and Murphy, A.S. Post-transcriptional regulation of auxin transport proteins: cellular trafficking, protein phosphorylation, protein maturation, ubiquitination, and membrane composition. Journal of Experimental Botany 60, 1093-1107, 2009.

Tsutsui, H., Karasawa, S., Okamura, Y. and Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nature Methods 5, 683-685, 2008.

van den Brûle, S. and Smart, C.C. The plant PDR family of ABC transporters. Planta 216, 95-106, 2002.

Verma, H.K., Shukla, P., Alfatah, M., Khare, A.K., Upadhyay, U., Ganesan, K. and Singh, J. High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast. Public Library of Science One 9, e101201, 2014.

Verrier, P.J., Bird, D., Burla, B., Dassa, E., Forestier, C., Geisler, M., Klein, M., Kolukisaoglu, Ü., Lee, Y. and Martinoia, E. Plant ABC proteins-a unified nomenclature and updated inventory. Trends in Plant Science 13, 151-159, 2008.

Vervecken, W., Callewaert, N., Kaigorodov, V., Geysens, S. and Contreras, R. Modification of the N-glycosylation pathway to produce homogeneous, human-like glycans using GlycoSwitch plasmids. Pichia Protocols, Springer, Germany, 119-138, 2007.

Vieira, S.M., da Rocha, S.L.G., Neves-Ferreira, A., Almeida, R.V. and Perales, J. Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris. Public Library of Science Neglected Tropical Diseases 11, e0005829, 2017.

Wachtler, V. and Balasubramanian, M.K. Yeast lipid rafts? -an emerging view. Trends in Cell Biology 16, 1-4, 2006.

Wachtler, V., Rajagopalan, S. and Balasubramanian, M.K. Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. Journal of Cell Science 116, 867-874, 2003.

Winzeler, E.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J.D. and Bussey, H. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901-906, 1999.

Worrall, D., Ng, C.K. and Hetherington, A.M. Sphingolipids, new players in plant signaling. Trends in Plant Science 8, 317-320, 2003.

Wu, M., Shen, Q., Yang, Y., Zhang, S., Qu, W., Chen, J., Sun, H. and Chen, S. Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115. Journal of Industrial Microbiology and Biotechnology 40, 589-599, 2013.

Xia, W.R., Fu, W.L., Cai, L., Cai, X., Wang, Y.Y., Zou, M.J. and Xu, D.G. Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris. Bioscience Biotechnology and Biochemistry 76, 1384-1388, 2012.

Yang, H. and Murphy, A.S. Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. The Plant Journal 59, 179-191, 2009.

Yang, H., Richter, G.L., Wang, X., Mlodzinska, E., Carraro, N., Ma, G., Jenness, M., Chao, D.Y., Peer, W.A. and Murphy, A.S. Sterols and sphingolipids differentially function in trafficking of the Arabidopsis ABCB19 auxin transporter. The Plant Journal 74, 37-47, 2013.

Yim, S., Khare, D., Kang, J., Hwang, J.U., Liang, W., Martinoia, E., Zhang, D., Kang, B. and Lee, Y. Postmeiotic development of pollen surface layers requires two Arabidopsis ABCG-type transporters. Plant Cell Reports 35, 1863-1873, 2016.

Zhan, H., Liu, B., Reid, S.W., Aoki, K.H., Li, C., Syed, R.S., Karkaria, C., Koe, G., Sitney, K. and Hayenga, K. Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin. Protein Engineering 12, 505-513, 1999.

Zhang, W., Zhao, F., Jiang, L., Chen, C., Wu, L. and Liu, Z. Different pathogen defense strategies in Arabidopsis: more than pathogen recognition. Cells 7, 252-276, 2018.

Zhu, M. and Fahl, W.E. Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents. Analytical Biochemistry 287, 210-217, 2000.