由實驗室先前的研究中，已知橙黃壺菌 BL10 的細胞能分泌一種蛋白質，來影響周遭細胞的分化及遷移。在將純化後的外泌蛋白利用 LC-MS/MS 進行定序後，與現有的 BL10 基因體資料庫比對的結果，發現有 5 個疑似能產生該蛋白的上游基因。本研究的目的為確認出何者為真正的上游基因，作法是將 5 個 candidate genes 利用 E. coli BL21 表現出重組蛋白，搭配後續的活性分析，找出何者具有活性。實驗結果證明這 5 個基因在 BL10 的對數成長初期後均會持續表現，且均可以利用E. coli製造出重組蛋白，然而在以陰離子交換層析及金屬離子親和層析進行純化時，這些重組蛋白均會難以如預期般附著於膠體上，以致難以純化。在將這 5 個經過純化的重組蛋白進行活性分析時，發現只有 #2034 和 #3413 具有近似於外泌蛋白般能降低阿米巴細胞比例的活性，其中更只有 #3413 具有近似於外泌蛋白般，對於阿米巴細胞具有化學排斥的活性，再加上 #3413 的推測胺基酸序列是5個candidate genes中最接近外泌蛋白者，據此推測 #3413 為該外泌蛋白上游基因的可能性很高。

Researchers have established that secretory proteins direct the differentiation and migration the Aurantiochytrium mangrovei strain BL10 cells. Our objective in this study was to identify the upstream gene associated with this bioactive protein. E. coli BL21 was used as an expression host to obtain recombinant proteins of five candidate genes (from the alignment of the LC-MS sequence of the bioactive protein and the genomic database of BL10). In activity assays, the recombinant proteins were compared with the activity of the secretory protein. Formation activity data revealed that #2034 and #3413 are associated with significant reductions in the proportion of amoeboid cells. In migration assays, only #3413 presented the chemorepellent properties of the bioactive protein. Amino acid sequences and activity analysis revealed that #3413 presented the strongest similarity to the bioactive protein. Thus, we speculate that #3413 is the upstream gene of the previously mentioned bioactive protein in BL10.

周思辰，探討橙黃壺菌BL10品系阿米巴細胞形成與遷移的機制，國立成功大學生物科技研究所碩士論文，2013。

郭宛靜，細菌性注射疫苗誘發石斑魚免疫因子基因表現及對保護力之影響，國立成功大學生物科技研究所碩士論文，2016。

陳宛渝，橙黃壺菌BL10品系之外泌蛋白純化與功能分析，國立成功大學生物科技研究所碩士論文，2015。

劉致寧，橙黃壺菌L-BL10品系之聚酮合成酶基因序列與表現分析，國立成功大學生物科技研究所碩士論文，2013。

蘇昱銘，來自北台灣之海洋異營性微藻分離株:Aurantiochytrium sp. strain BL10之生物特性研究，國立成功大學生物科技研究所碩士論文，2012。

Abbas, S.S., Naveed, S., Qamar, F. and Ali, Z.B. Enlightenment on naegleria fowleri; a brain eating amoeba-an alarming call. International Education and Research Journal 1, 1-3, 2015.

Aebi, M. N-linked protein glycosylation in the ER. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1833, 2430-2437, 2013.

Anjard, C., van Bemmelen, M., Véron, M. and Reymond, C.D. A new spore differentiation factor (SDF) secreted by Dictyostelium cells is phosphorylated by the cAMP dependent protein kinase. Differentiation 62, 43-49, 1997.

Bornhorst, J.A. and Falke, J.J. Purification of proteins using polyhistidine affinity tags. In Methods in Enzymology 326, 245-254, 2000.

Bosgraaf, L., and Van Haastert, P.J. The ordered extension of pseudopodia by amoeboid cells in the absence of external cues. Public Library of Science One 4, e5253, 2009.

Brock, D.A., and Gomer, R.H. A secreted factor represses cell proliferation in Dictyostelium. Development 132, 4553-4562, 2005.
Chaung, K. C., Chu, C. Y., Su, Y. M. and Chen, Y. M. Effect of culture conditions on growth, lipid content, and fatty acid composition of Aurantiochytrium mangrovei strain BL10. Applied Microbiology and Biotechnology Express 2, 42, 2012.

Corfield, A. Eukaryotic protein glycosylation: a primer for histochemists and cell biologists. Histochemistry and Cell Biology 147, 119-147, 2017.

Darby, I.A., Laverdet, B., Bonté, F. and Desmoulière, A. Fibroblasts and myofibroblasts in wound healing. Clinical, Cosmetic and Investigational Dermatology 7, 301, 2014.

Debeljak, N., Feldman, L., Davis, K.L., Komel, R. and Sytkowski, A.J. Variability in the immunodetection of His-tagged recombinant proteins. Analytical Biochemistry 359, 216-223, 2006.

Foxman, E.F., Kunkel, E.J. and Butcher, E.C. Integrating Conflicting Chemotactic Signals: The role of memory in leukocyte navigation. The Journal of Cell Biology 147, 577-588, 1999.

Friedl, P. and Wolf, K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Reviews Cancer 3, 362, 2003.

Gustafsson, C., Govindarajan, S. and Minshull, J. Codon bias and heterologous protein expression. Trends in Biotechnology 22, 346-353, 2004.

Harvath, L. Neutrophil chemotactic factors. Cell Motility Factors 59, 35-52. 1991.

Imai, F., Sawada, M., Suzuki, H., Kiya, N., Hayakawa, M., Nagatsu, T., Marunouchi, T. and Kanno, T. Migration activity of microglia and macrophages into rat brain. Neuroscience Letters 237, 49-52, 1997.

Kamakura, S., Nomura, M., Hayase, J., Iwakiri, Y., Nishikimi, A., Takayanagi, R., Fukui, Y. and Sumimoto, H. The Cell Polarity Protein mInsc Regulates Neutrophil Chemotaxis via a Noncanonical G Protein Signaling Pathway, Developmental Cell 26, 292-302, 2013.
Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Current Opinion in Biotechnology 6, 494-500, 1995.

Kesbeke, F., HAASTERT, P.J., De Wit, R.J. and Snaar-Jagalska, B.E. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum. Journal of Cell Science 96, 668-673, 1990.

Lewis, T.E., Nichols, P.D. and McMeekin, T.A. The biotechnological potential of thraustochytrids. Marine Biotechnology 1, 580-587, 1999.

Li, A., Kato, Z., Ohnishi, H., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y. and Kondo, N. Optimized gene synthesis and high expression of human interleukin-18. Protein Expression and Purification 32, 110-118, 2003.

Medus, M.L., Gomez, G.E., Zacchi, L.F., Couto, P.M., Labriola, C.A., Labanda, M.S., Bielsa, R.C., Clérico, E.M., Schulz, B.L. and Caramelo, J.J. N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins. Scientific Reports 7, 8788, 2017.

Montana, V. Glioma: the mechanisms of infiltrative growth. Opera Medica et Physiologica 1, 69-76, 2016.

Morris, H.R., Taylor, G.W., Masento, M.S., Jermyn, K.A. and Kay, R.R. Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum. Nature 328, 811, 1987.

Nishigami, Y., Ichikawa, M., Kazama, T., Kobayashi, R., Shimmen, T., Yoshikawa, K. and Sonobe, S. Reconstruction of active regular motion in amoeba extract: Dynamic cooperation between sol and gel states. Public Library of Science One 8, e70317, 2013.

Oohata, A.A., Nakagawa, M., Tasaka, M. and Fujii, S. A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells. Development 124, 2781-2787, 1997.

Orgaz, J.L., Pandya, P., Dalmeida, R., Karagiannis, P., Sanchez-Laorden, B., Viros, A., Albrengues, J., Nestle, F.O., Ridley, A.J. and Gaggioli, C. Diverse matrix metalloproteinase functions regulate cancer amoeboid migration. Nature Communications 5, 4255, 2014.

Phillips, J.E. and Gomer, R.H. A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum. Proceedings of the National Academy of Sciences 109, 10990-10995, 2012.

Ralph, P.J. and Short, F.T. Impact of the wasting disease pathogen, Labyrinthula zosterae, on the photobiology of eelgrass Zostera marina. Marine Ecology Progress Series 226, 265-271, 2002.

Seifert, K. N., Adderson, E. E., Whiting, A. A., Bohnsack, J. F., Crowley, P. J. and Brady, L. J. A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ε) associated with a virulent lineage of serotype III Streptococcus agalactiae. Microbiology 152, 1029-1040, 2006.

Sinclair, A.M. and Elliott, S. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. Journal of Pharmaceutical Sciences 94, 1626-1635, 2005.

Smirnova, T. and Segall, J.E. Amoeboid chemotaxis: future challenges and opportunities. Cell Adhesion and Migration 1, 165-170, 2007.

Solá, R.J. and Griebenow, K. Effects of glycosylation on the stability of protein pharmaceuticals. Journal of Pharmaceutical Sciences 98, 1223-1245, 2009.

Strassmann, J.E., Zhu, Y. and Queller, D.C. Altruism and social cheating in the social amoeba Dictyostelium discoideum. Nature 408, 965, 2000.

Strittmatter, M., Guerra, T., Silva, J. and Gachon, C.M. A new flagellated dispersion stage in Paraphysoderma sedebokerense, a pathogen of Haematococcus pluvialis. Journal of Applied Phycology 28, 1553-1558, 2016.

Takao, Y., Nagasaki, K., Mise, K., Okuno, T. and Honda, D. Isolation and characterization of a novel single-stranded RNA virus infectious to a marine fungoid protist, Schizochytrium sp.(Thraustochytriaceae, Labyrinthulea). Applied and Environmental Microbiology 71, 4516-4522, 2005.
Takegawa, K., Tohda, H., Sasaki, M., Idiris, A., Ohashi, T., Mukaiyama, H., Giga‐Hama, Y. and Kumagai, H. Production of heterologous proteins using the fission‐yeast (Schizosaccharomyces pombe) expression system. Biotechnology and Applied Biochemistry 53, 227-235, 2009.

Vallejo, L.F. and Rinas, U. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories 3, 11, 2004.

Vogel, G., Thilo, L., Schwarz, H. and Steinhart, R. Mechanism of phagocytosis in Dictyostelium discoideum: phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties. The Journal of Cell Biology 86, 456-465, 1980.

Wahid, M.I., Yoshikawa, T. and Sakata, T. Bacteriolytic activity and growth of marine isolates of labyrinthulids on dead bacterial cells. Fisheries Science 73, 1286-1294, 2007.

Xinyao, L., Miao, S., Yonghong, L., Yin, G., Zhongkai, Z., Donghui, W., Weizhong, W. and Chencai, A. Feeding Characteristics of an Amoeba (Lobosea: Naegleria) Grazing Upon Cyanobacteria: Food Selection, Ingestion and Digestion Progress. Microbial Ecology 51, 315-325, 2006.

Yang, H. L., Lu, C. K., Chen, S. F., Chen, Y. M. and Chen, Y. M. Isolation and Characterization of Taiwanese Heterotrophic Microalgae: Screening of Strains for Docosahexaenoic Acid (DHA) Production. Marine Biotechnology 12, 173-185, 2010.

Zhu, S., Gong, C., Ren, L., Li, X., Song, D. and Zheng, G. A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression. Applied Microbiology and Biotechnology 97, 837-845, 2013.