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

• Main Authors: Hao-An Hsiung, 熊浩安
• Other Authors: 黃慶璨
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/9294e2

碩士 === 國立臺灣大學 === 生化科技學系 === 107 === 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.