請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82429完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Chun-Fu Yu | en |
| dc.contributor.author | 游鈞富 | zh_TW |
| dc.date.accessioned | 2022-11-25T07:30:53Z | - |
| dc.date.available | 2023-09-14 | |
| dc.date.copyright | 2021-11-06 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-15 | |
| dc.identifier.citation | 1. M.D. Binder, N. Hirokawa, and U. Windhorst, Heterologous Expression, in Encyclopedia of Neuroscience, SpringerLink. 2009. p. 1830-1830. 2. Gomes, A.R., et al., An overview of heterologous expression host systems for the production of recombinant proteins. Adv Anim Vet Sci, 2016. 4: p. 346-356. 3. Gellissen, G., Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol, 2000. 54(6): p. 741-50. 4. Cregg, J.M., et al., Recombinant protein expression in Pichia pastoris. Mol Biotechnol, 2000. 16(1): p. 23-52. 5. Ahmad, M., et al., Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol, 2014. 98(12): p. 5301-17. 6. Damasceno, L.M., et al., Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, 2012. 93(1): p. 31-9. 7. Guilliermond, A., Zygosaccharomyces Pastori, nouvelle espèce de levures à copulation hétérogamique. 1920. 8. Phaff, H.J., M.W. Miller, and M. Shifrine, The taxonomy of yeasts isolated from Drosophila in the Yosemite region of California. Antonie van Leeuwenhoek, 1956. 22(1): p. 145-161. 9. Wegner, G.H. and W. Harder, Methylotrophic yeasts-1986. Microbial Growth on C1 Compounds, 1987. p. 131-138. 10. Yamada, Y., et al., The phylogenetic relationships of methanol-assimilating yeasts based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Komagataella gen. nov. (Saccharomycetaceae). Biosci Biotechnol Biochem, 1995. 59(3): p. 439-44. 11. Kurtzman, C.P., Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J Ind Microbiol Biotechnol, 2009. 36(11): p. 1435-8. 12. Naumov, G.I., E.S. Naumova, and K.L. Boundy-Mills, Description of Komagataella mondaviorum sp. nov., a new sibling species of Komagataella (Pichia) pastoris. Antonie Van Leeuwenhoek, 2018. 111(7): p. 1197-1207. 13. Bill, R.M., Yeast-a panacea for the structure-function analysis of membrane proteins? Curr Genet, 2001. 40(3): p. 157-71. 14. Cregg, J.M., T.S. Vedvick, and W.C. Raschke, Recent Advances in the Expression of Foreign Genes in Pichia pastoris. Nat Biotechnol, 1993. 11(8): p. 905-910. 15. Waters, M.G., E.A. Evans, and G. Blobel, Prepro-alpha-factor has a cleavable signal sequence. J Biol Chem, 1988. 263(13): p. 6209-14. 16. Bevan, A., C. Brenner, and R.S. Fuller, Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system. Proc Natl Acad Sci U S A, 1998. 95(18): p. 10384-9. 17. Wagner, J.C. and D.H. Wolf, Hormone (pheromone) processing enzymes in yeast. The carboxy-terminal processing enzyme of the mating pheromone alpha-factor, carboxypeptidase ysc alpha, is absent in alpha-factor maturation-defective kex1 mutant cells. FEBS Lett, 1987. 221(2): p. 423-6. 18. Lin-Cereghino, G.P., et al., The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene, 2013. 519(2): p. 311-7. 19. Barrero, J.J., et al., An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact, 2018. 17(1): p. 161. 20. Fitzgerald, I. and B.S. Glick, Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting. Microb Cell Fact, 2014. 13(1): p. 125. 21. Zhang, W., et al., Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A. Biotechnol Bioeng, 2000. 70(1): p. 1-8. 22. Sinha, J., et al., Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. Biotechnol Bioeng, 2005. 89(1): p. 102-12. 23. Sahu, U., K. Krishna Rao, and P.N. Rangarajan, Trm1p, a Zn(II)₂Cys₆-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris. Biochem Biophys Res Commun, 2014. 451(1): p. 158-64. 24. Cámara, E., et al., Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris. Sci Rep, 2017. 7: p. 44302. 25. Chang, C.H., et al., Enhancing the efficiency of the Pichia pastoris AOX1 promoter via the synthetic positive feedback circuit of transcription factor Mxr1. BMC Biotechnol, 2018. 18(1): p. 81. 26. Anelli, T. and R. Sitia, Protein quality control in the early secretory pathway. Embo j, 2008. 27(2): p. 315-27. 27. Ellgaard, L. and A. Helenius, Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol, 2003. 4(3): p. 181-191. 28. Dobson, C.M., Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol, 2004. 15(1): p. 3-16. 29. Gasser, B., et al., Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact, 2008. 7: p. 11. 30. Idiris, A., et al., Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol, 2010. 86(2): p. 403-17. 31. Delic, M., et al., Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins. Antioxid Redox Signal, 2014. 21(3): p. 414-37. 32. Hwang, J. and L. Qi, Quality Control in the Endoplasmic Reticulum: Crosstalk between ERAD and UPR pathways. Trends Biochem Sci, 2018. 43(8): p. 593-605. 33. Chakraborty, R., et al., Comparison and contrast of plant, yeast, and mammalian ER stress and UPR. Applied Biological Chemistry, 2016. 59(3): p. 337-347. 34. Chakrabarti, A., A.W. Chen, and J.D. Varner, A review of the mammalian unfolded protein response. Biotechnol Bioeng, 2011. 108(12): p. 2777-93. 35. Wu, H., B.S. Ng, and G. Thibault, Endoplasmic reticulum stress response in yeast and humans. Biosci Rep, 2014. 34(4). 36. Cox, J.S., C.E. Shamu, and P. Walter, Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell, 1993. 73(6): p. 1197-206. 37. Walter, P. and D. Ron, The unfolded protein response: from stress pathway to homeostatic regulation. Science, 2011. 334(6059): p. 1081-6. 38. Pincus, D., et al., BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response. PLoS Biol, 2010. 8(7): p. e1000415. 39. Bertolotti, A., et al., Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol, 2000. 2(6): p. 326-32. 40. Okamura, K., et al., Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. Biochem Biophys Res Commun, 2000. 279(2): p. 445-50. 41. Kimata, Y., et al., A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1. J Cell Biol, 2004. 167(3): p. 445-56. 42. Mori, K., et al., A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell, 1993. 74(4): p. 743-56. 43. Shamu, C.E. and P. Walter, Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. Embo j, 1996. 15(12): p. 3028-39. 44. Sidrauski, C. and P. Walter, The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell, 1997. 90(6): p. 1031-9. 45. Rubio, C., et al., Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity. J Cell Biol, 2011. 193(1): p. 171-84. 46. Cox, J.S. and P. Walter, A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell, 1996. 87(3): p. 391-404. 47. Mori, K., et al., Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells, 1996. 1(9): p. 803-17. 48. Kawahara, T., et al., Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol Biol Cell, 1997. 8(10): p. 1845-62. 49. Rüegsegger, U., J.H. Leber, and P. Walter, Block of HAC1 mRNA translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response. Cell, 2001. 107(1): p. 103-14. 50. Whyteside, G., et al., Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p. FEBS Lett, 2011. 585(7): p. 1037-41. 51. Mori, K., et al., A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. Embo J, 1992. 11(7): p. 2583-93. 52. Travers, K.J., et al., Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell, 2000. 101(3): p. 249-58. 53. Damasceno, L.M., et al., Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris. Appl Microbiol Biotechnol, 2007. 74(2): p. 381-9. 54. Vad, R., et al., Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol, 2005. 116(3): p. 251-60. 55. Lodish, H.F., et al., Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature, 1983. 304(5921): p. 80-3. 56. Shusta, E.V., et al., Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol, 1998. 16(8): p. 773-7. 57. Parekh, R., K. Forrester, and D. Wittrup, Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae. Protein Expr Purif, 1995. 6(4): p. 537-45. 58. Shuster, J.R., Gene expression in yeast: protein secretion. Curr Opin Biotechnol, 1991. 2(5): p. 685-90. 59. Matlack, K.E., et al., BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane. Cell, 1999. 97(5): p. 553-64. 60. Rowling, P.J. and R.B. Freedman, Folding, assembly, and posttranslational modification of proteins within the lumen of the endoplasmic reticulum. Subcell Biochem, 1993. 21: p. 41-80. 61. Wilkinson, B. and H.F. Gilbert, Protein disulfide isomerase. Biochim Biophys Acta, 2004. 1699(1-2): p. 35-44. 62. Ng, D.T., J.D. Brown, and P. Walter, Signal sequences specify the targeting route to the endoplasmic reticulum membrane. Journal of Cell Biology, 1996. 134(2): p. 269-278. 63. Plath, K., et al., Signal Sequence Recognition in Posttranslational Protein Transport across the Yeast ER Membrane. Cell, 1998. 94(6): p. 795-807. 64. Rakestraw, J.A., et al., Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2009. 103(6): p. 1192-1201. 65. Willer, M., G.M.A. Forte, and C.J. Stirling, Sec61p Is Required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY. J Biol Chem, 2008. 283(49): p. 33883-33888. 66. Forte, G.M.A., M.R. Pool, and C.J. Stirling, N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum. PLoS Biol, 2011. 9(5): p. e1001073. 67. Hackel, B.J., et al., Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res, 2006. 23(4): p. 790-797. 68. Li, Z., et al., Low-Temperature Increases the Yield of Biologically Active Herring Antifreeze Protein in Pichia pastoris. Protein Expr Purif, 2001. 21(3): p. 438-445. 69. Lin, H., et al., Enhancing the production of Fc fusion protein in fed‐batch fermentation of Pichia pastoris by design of experiments. Biotechnol Prog, 2007. 23(3): p. 621-625. 70. Shi, X., et al., Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif, 2003. 28(2): p. 321-330. 71. Gasser, B., et al., Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics, 2007. 8(1): p. 179. 72. Smith, J.D., N.E. Richardson, and A.S. Robinson, Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress. Biochim et Biophys Acta Proteins Proteom, 2005. 1752(1): p. 18-25. 73. Bull, R.A., M.M. Tanaka, and P.A. White, Norovirus recombination. J Gen Virol, 2007. 88(Pt 12): p. 3347-3359. 74. Li, J., et al., New interventions against human norovirus: progress, opportunities, and challenges. Annu Rev Food Sci Technol, 2012. 3: p. 331-52. 75. Chen, Y.L., P.J. Chang, and C.T. Huang, Small P particles formed by the Taiwan-native norovirus P domain overexpressed in Komagataella pastoris. Appl Microbiol Biotechnol, 2018. 102(22): p. 9707-9718. 76. Conly, J. and B. Johnston, Norwalk virus - Off and running. Can J Infect Dis, 2003. 14(1): p. 11-3. 77. Moore, M.D., R.M. Goulter, and L.A. Jaykus, Human norovirus as a foodborne pathogen: challenges and developments. Annu Rev Food Sci Technol, 2015. 6: p. 411-33. 78. Lopman, B.A., et al., The Vast and Varied Global Burden of Norovirus: Prospects for Prevention and Control. PLoS Med, 2016. 13(4): p. e1001999. 79. Koopmans, M., Progress in understanding norovirus epidemiology. Curr Opin Infect Dis, 2008. 21(5): p. 544-52. 80. Bayer, M.E., B.S. Blumberg, and B. Werner, Particles associated with Australia antigen in the sera of patients with leukaemia, Down's Syndrome and hepatitis. Nature, 1968. 218(5146): p. 1057-9. 81. Serino, L., Seib, L.,and Pizza, M., Design of New Vaccines in Genomic and Post-genomic Era, in Innovation in Vaccinology: from design, through to delivery and testing, S. Baschieri, Editor. 2012, Springer Netherlands: Dordrecht. p. 3-15. 82. Grimm, S.K. and M.E. Ackerman, Vaccine design: emerging concepts and renewed optimism. Curr Opin Biotechnol, 2013. 24(6): p. 1078-88. 83. Rebeaud, F. and M. Bachmann, Virus-Like Particles as Efficient Delivery Platform to Induce a Potent Immune Response, in Innovation in Vaccinology: from design, through to delivery and testing, S. Baschieri, Editor. 2012, Springer Netherlands: Dordrecht. p. 87-122. 84. Atmar, R.L., S. Ramani, and M.K. Estes, Human noroviruses: recent advances in a 50-year history. Curr Opin Infect Dis, 2018. 31(5): p. 422-432. 85. Prasad, B.V., et al., X-ray crystallographic structure of the Norwalk virus capsid. Science, 1999. 286(5438): p. 287-90. 86. Greenberg, H.B., et al., Proteins of Norwalk virus. J Virol, 1981. 37(3): p. 994-999. 87. Hardy, M.E., et al., Specific proteolytic cleavage of recombinant Norwalk virus capsid protein. J Virol, 1995. 69(3): p. 1693-1698. 88. Fang, H., et al., Norovirus P particle efficiently elicits innate, humoral and cellular immunity. PLoS One, 2013. 8(4): p. e63269. 89. Bárcena, J. and E. Blanco, Design of novel vaccines based on virus-like particles or chimeric virions. Subcell Biochem, 2013. 68: p. 631-65. 90. Tan, M., et al., Norovirus P particle, a novel platform for vaccine development and antibody production. J Virol, 2011. 85(2): p. 753-64. 91. Tan, M. and X. Jiang, Norovirus P particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus. Nanomedicine (Lond), 2012. 7(6): p. 889-97. 92. Xia, M., et al., A candidate dual vaccine against influenza and noroviruses. Vaccine, 2011. 29(44): p. 7670-7. 93. Fu, L., et al., Characterization of NoV P particle-based chimeric protein vaccines developed from two different expression systems. Protein Expr Purif, 2017. 130: p. 28-34. 94. Fu, L., et al., Norovirus P particle-based active Aβ immunotherapy elicits sufficient immunogenicity and improves cognitive capacity in a mouse model of Alzheimer's disease. Sci Rep, 2017. 7: p. 41041. 95. Li, Y., et al., Establishment of a novel method without sequence modification for developing NoV P particle-based chimeric vaccines. Protein Expr Purif, 2016. 121: p. 73-80. 96. Hsieh, S.-Y., Enhancing AOX1 promoter efficiency of Konagataella phaffii (Pichia pastoris) using CRISPRi and CRISPRa. National Taiwan University Master Thesis, 2020. 97. Hsiung, H.-A., Effects of Additional Ire1 Expression on Secretory Efficiency of Anti-EGFR ScFv in Pichia pastoris. National Taiwan University Master Thesis, 2018. 98. Huang, M., et al., Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris. Bioprocess Biosyst Eng, 2017. 40(3): p. 341-350. 99. Guerfal, M., et al., The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact, 2010. 9: p. 49. 100. Yang, J., et al., Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris. Appl Microbiol Biotechnol, 2016. 100(12): p. 5453-65. 101. Meusser, B., et al., ERAD: the long road to destruction. Nat Cell Biol, 2005. 7(8): p. 766-72. 102. Carvalho, N.D.S.P., et al., Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger. Appl Microbiol Biotechnol, 2011. 89(2): p. 357-373. 103. Belden, W.J. and C. Barlowe, Deletion of yeast p24 genes activates the unfolded protein response. Mol Biol Cell, 2001. 12(4): p. 957-69. 104. Liu, Y.Y., J.H. Woo, and D.M. Neville, Jr., Overexpression of an anti-CD3 immunotoxin increases expression and secretion of molecular chaperone BiP/Kar2p by Pichia pastoris. Appl Environ Microbiol, 2005. 71(9): p. 5332-40. 105. Saraste, J., G.E. Palade, and M.G. Farquhar, Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci, 1986. 83(17): p. 6425-6429. 106. Dragosits, M., et al., The Effect of Temperature on the Proteome of Recombinant Pichia pastoris. J Proteome Res, 2009. 8(3): p. 1380-1392. 107. Zepeda, A., A. Pessoa, and J. Farias, Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast. Braz J Microbiol, 2018. 49 Suppl 1. 108. R S Fuller, a. R E Sterne, and J. Thorner, Enzymes Required for Yeast Prohormone Processing. Annu Rev Physiol, 1988. 50(1): p. 345-362. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82429 | - |
| dc.description.abstract | "畢赤氏酵母菌 Pichia pastoris 為廣泛應用的異源蛋白質表達系統，兼具單一細胞與真核生物的特點，其優勢為生長快速、培養成本低廉並可進行高密度培養，且具有適當的轉譯後修飾及外泌重組蛋白質的能力。然而，P. pastoris 經誘導大量生產異源蛋白質時，往往會因為內質網的折疊效率不足，導致過多的未折疊蛋白質累積於內質網中，造成內質網壓力 (endoplasmic reticulum stress)，且促使未折疊蛋白質反應 (unfolded protein response, UPR) 發生。位於內質網膜上的 Ire1 (inositol-requiring enzyme 1) 受內質網壓力影響，會活化自身的核酸內切酶活性，切除轉錄因子 HAC1 mRNA 的內含子。藉此調控其下游基因，協助蛋白質折疊外泌；或促使蛋白質降解來舒緩內質網壓力。 本研究以 E2-Crimson 四聚體紅色螢光蛋白質作為模式蛋白質，發現甲醇誘導 27 小時後，額外表現轉錄因子 Hac1 之菌株其 HAC1 基因 mRNA 相對表現量提升了 14.8 倍。觀察螢光量發現，額外表現 Hac1 使 E2-Crimson 的外泌量提升了 1.6 倍。UPR 之相關基因 KAR2 的 mRNA 相對表現量提升，為 1.45 倍；內質網相關蛋白質降解途徑 (ER-associated protein degradation) 之相關基因 HRD1 及 UBC1 的 mRNA 相對表現量則降低，分別為 0.64 倍及 0.47 倍。進一步將此策略應用於諾羅病毒 P 蛋白質的外泌生產。P 粒子 (P particle) 是由諾羅病毒衣殼蛋白質中的突出區 (protruding domain) 組成，可用於生產類病毒顆粒疫苗；具有多抗原呈現的功能，可作為疫苗發展平台。本實驗室在前人 P. pastoris 胞內表現諾羅病毒 P 蛋白質的基礎下，已成功地將諾羅病毒 P 蛋白質外泌至胞外。本研究希望藉由額外表現轉錄因子 Hac1 的方式提升諾羅病毒 P 蛋白質之外泌效果，讓其於回收及純化上更有效率，增加 P. pastoris 表現系統的應用性。目前已成功證實額外表現 Hac1 可提升以 P. pastoris 生產諾羅病毒 P 蛋白質的外泌效率。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T07:30:53Z (GMT). No. of bitstreams: 1 U0001-1409202113100200.pdf: 3263074 bytes, checksum: 16b39839318f0e2f10651d3f709a469e (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 摘要 I Abstract III 圖目錄 VIII 表目錄 IX 第一章 前言 1 一、 異源蛋白質表現系統 1 二、 Pichia pastoris 表現系統 2 1. Pichia pastoris 被重新分類成 Komagataella phaffii 2 2. 表現強力且可受甲醇調控的 PAOX1 啟動子 3 3. 真核表現系統的優勢 3 4. 蛋白質外泌系統 4 三、 提升 PAOX1 表現強度的轉錄調控策略 5 四、 P. pastoris 表現系統之外泌瓶頸 6 1. 未摺疊蛋白質反應 6 2. 內質網相關蛋白質降解 7 五、 提升外泌效率的策略 8 1. 額外表現蛋白質摺疊相關基因 8 2. 外泌訊號序列 9 3. 調整誘導溫度 9 六、 諾羅病毒 P 蛋白質 10 1. 諾羅病毒 10 2. 類病毒顆粒疫苗 10 3. 多抗原呈現平台 11 七、 研究動機 12 1. 研究策略 14 2. 研究目標 15 第二章 材料與方法 16 一、 實驗菌株與培養條件 16 1. 細菌 16 2. 真菌 16 3. 菌株保存 16 二、 培養基 16 三、 表現載體建構 19 四、 嗜甲醇酵母菌電穿孔轉形 22 1. P. pastoris 勝任細胞製備 22 2. 電穿孔轉形 22 五、 轉型株之抗性篩選 23 六、 Pichia pastoris 轉形株培養與分析 23 1. 96 深孔盤誘導 23 2. 搖瓶誘導 23 3. 醱酵槽生產 24 七、 mRNA 表現量分析 25 八、 蛋白質分析 27 1. 十二烷基硫酸鈉聚丙烯醯胺膠體電泳 (SDS-PAGE) 27 2. 西方墨點法 27 3. 螢光強度分析 28 4. Bradford 蛋白質定量法 28 5. 酵素結合免疫吸附分析法 28 第三章 結果 32 一、 取得 HAC1 基因編碼序列 32 二、 額外表現 Hac1 對外泌 E2-Crimson 菌株的影響 34 三、 額外表現 Hac1 提升諾羅病毒 P 蛋白質之外泌產量 42 四、 使用醱酵槽大量生產諾羅病毒 P 蛋白質 48 第四章 討論 54 一、 P. pastoris 表現系統外泌效率之瓶頸 54 二、 額外表現 Hac1 之菌株面對內質網壓力時的反應 55 三、 額外表現 Hac1 對菌株外泌目標蛋白質之影響 57 第五章 結論 59 第六章 未來展望 60 第七章 參考文獻 62 | |
| dc.language.iso | zh-TW | |
| dc.title | 以額外表達 Hac1 策略增強 Komagataella phaffii (Pichia pastoris) 外泌效率 | zh_TW |
| dc.title | Enhancement of Secretory Efficiency by Additional Expression of Hac1 in Komagataella phaffii (Pichia pastoris) | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林玉儒(Hsin-Tsai Liu),傅煦媛(Chih-Yang Tseng) | |
| dc.subject.keyword | Pichia pastoris,外泌,Hac1,異源蛋白質生產,未摺疊蛋白質, | zh_TW |
| dc.subject.keyword | Pichia pastoris,Secretion,Hac1,Heterologous protein production,Unfolded protein response, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202103163 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-09-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2023-09-14 | - |
| 顯示於系所單位： | 生化科技學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-1409202113100200.pdf | 3.19 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。