額外表現Ire1對Pichia pastoris外泌生產抗EGFR單鏈抗體之影響

Effects of Additional Ire1 Expression on Secretory Efficiency of Anti-EGFR ScFv in Pichia pastoris

熊浩安(Hao-An Hsiung)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/生化科技學系/碩士(2018年)

https://doi.org/10.6342/NTU201804143

摘要

Pichia pastoris 為嗜甲醇酵母菌的一種，兼具微生物與真核系統的優勢，如培養成本低廉、生長快速，並具有轉譯後修飾、蛋白質外泌能力，利用甲醇誘導的AOX1啟動子，可大量生產重組蛋白質，是極具潛力的異源蛋白質表達系統。然而，生產異源外泌蛋白質時，大量的蛋白質常因內質網摺疊效率不足而導致未摺疊蛋白質的累積，限制蛋白質的外泌，並在細胞內產生內質網壓力 (ER stress) 進而引發未摺疊蛋白質反應 (unfolded protein response, UPR)。P. pastoris的未摺疊蛋白質反應主要由內質網內的Ire1感應，並藉由切除轉錄因子HAC1 mRNA的內含子使之轉譯出有功能的蛋白質，藉此調控下游協助蛋白質摺疊及將蛋白質降解等反應以解除內質網壓力。

本論文以P. pastoris生產表皮生長因子受體 (epidermal growth factor receptor, EGFR) 之單鏈抗體 (single-chain variable fragment, scFv) 時，發現以前人論文中的轉錄因子Mxr1再程序化策略表現目標蛋白質時，外泌效率可能是生產的瓶頸。推測P. pastoris內生的未摺疊蛋白質反應不足以疏通外泌路徑的阻塞。因此本論文希望透過一個同樣需甲醇誘導但強度較低的AOX2啟動子額外提升Ire1的表現，增強細胞內的未摺疊蛋白反應，以更有效率地處理過多的目標蛋白質累積，增加蛋白質正確摺疊及外泌效率。並研究額外表現Ire1對細胞的影響，包含目標蛋白質的表現量、未摺疊蛋白質反應的下游路徑調控等。結果顯示，額外表現Ire1同時提升了協助蛋白質摺疊及將蛋白質降解兩種反應，使胞內累積的目標蛋白質明顯減少，但胞外蛋白質產量並無顯著提升，顯示Ire1可能還有其他未被發現的調控路徑，期望透過此研究更加釐清P. pastoris未摺疊蛋白質反應的調控路徑，未來有機會將P. pastoris發展為更具外泌生產效率的蛋白質生產系統，增進其應用性。

關鍵字

Pichia pastoris ； 未摺疊蛋白質反應 ； Mxr1 ； Ire1 ； 單鏈抗體

並列摘要

Pichia pastoris has been successfully applied in the production of many recombinant proteins because of its distinguished features that combine the advantages of both microbial and eukaryotic system. Moreover, recombinant proteins can be secreted to the medium through addition of a secretory signal sequence, which simplifies the purification process. In P. pastoris, recombinant proteins production was usually driven by methanol-induced AOX1 promoter because of its strong characteristics. However, owing to the high-level expression of recombinant proteins, secretory process such as protein folding within the ER tends to be the most important limitation which reduce the productivity. The accumulation of unfolded proteins in the ER will induce a process known as unfolded protein response (UPR) that can help reduce the stress. UPR is activated by Inositol-requiring enzyme–1 (Ire1) through the translation of transcription factor Hac1. This response can relieve the ER stress by the expression of genes involved in protein folding, modification, translocation. In this research, anti-epidermal growth factor receptor (EGFR) scFv is produced in P. pastoris. Although the efficiency of AOX1 promoter was enhanced by reprogramming the transcription factor Mxr1, the recombinant proteins could not be secreted efficiently. Therefore, additional Ire1 expression driven by AOX2 promoter was applied to enhance the secretory capacity by UPR activation. Both UPR-responsive genes and genes related to ER-associated degradation were upregulated by extra Ire1. Besides, intracellular scFv was eliminated. These results suggested that there might be other signaling pathway regulated by Ire1. Further experiments are needed to demonstrate the feasibility of additional Ire1 expression in the production of recombinant proteins in Pichia pastoris.

並列關鍵字

Pichia pastoris ； unfolded protein response ； Mxr1 ； Ire1 ； scFv

參考文獻

1. Gellissen, G. (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54, 741-750

2. Cregg, J. M., Cereghino, J. L., Shi, J., and Higgins, D. R. (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16, 23-52

3. Ahmad, M., Hirz, M., Pichler, H., and Schwab, H. (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98, 5301-5317

4. Damasceno, L. M., Huang, C. J., and Batt, C. A. (2012) Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93, 31-39

5. Zhang, W., Bevins, M. A., Plantz, B. A., Smith, L. A., and Meagher, M. M. (2000) 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 70, 1-8