利用螢光流式細胞分選儀於嗜甲醇酵母菌Pichia pastoris建立高通量篩選系統

Abstract

Pichia pastoris為嗜甲醇酵母菌的一種，是常用的異源蛋白質表達系統，具低生產成本、轉譯後修飾能力、能利用甲醇調控蛋白質生產等特色。傳統上高表現量的P. pastoris轉形株是利用抗生素濃度梯度篩選，但此方式相當地耗時費力且效果不彰。而目前使用96孔培養盤，以小量培養基同時培養多株轉形株，再分析蛋白質表現量，但此法使用的培養基體積太小，導致其中的轉形株活性容易受培養基內養份、廢物等的影響，導致其篩選的結果不一定是菌株本身能力優劣，而是因為培養基環境好壞的影響。本研究欲建立一個穩定的高通量篩選P. pastoris高表現量轉形株系統，方法為參考前人研究，建構一個多基因表現載體，利用來自口蹄疫病毒 (Foot and mouth disease virus) 中可進行自我截切的2A胜肽基因 (2A peptide gene)，連接目標蛋白質基因 (使用Mouse Endostatin-IL2融合蛋白作為目標蛋白質) 與綠色螢光蛋白質基因 (Enhance green fluorescent protein, EGFP)。利用綠色螢光強度間接代表目標蛋白質的表現量，並藉由螢光流式細胞分選儀 (Fluorescent-activated cell sorter, FACS)篩選具有高螢光表現量的轉形株。此外，前人使用不須後轉譯修飾的原核細胞蛋白質作為目標，因此本研究將目標置換成需後轉譯修飾，構形較為複雜的真核細胞融合蛋白質，結果顯示，本系統能夠順利表現真核細胞蛋白質。此外，轉形株的綠色螢光強度和目標蛋白質表現量呈現正相關，但由於使用單一菌落作為篩選材料，導致藉由螢光流式細胞分選儀篩選出來的轉形株，其表現量未如預期的提升。本研究確立了綠色螢光強度能作為目標蛋白質表現量的預測指標，但欲利用此系統篩選出高表現量轉形株，則仍需繼續研究。

The methylotrophic yeast Pichia pastoris is a generally used expression system in production of industrial and pharmaceutical proteins. Traditionally, screening high-yield candidates relies on assay transformant by transformant. However, It is labor extensive, time consuming and unstable. In this study, a high-throughput screening system in P. pastoris was developed. The polycistronic expression plasmids harboring the genes of Endostatin-IL2, 2A peptides from foot-and-mouth disease virus and EGFP were introduced into P. pastoris. The transformants and wild types were distinguished by their fluorescence. Furthermore, since previous study using prokaryotic protein as target protein, this study replace the protein to the eukaryotic fusion protein with more complex structure. The results showed this system successfully express the eukaryotic fusion protein. There is positive correlation between the production of Endo-IL2 and EGFP. Furthermore, the transformants could be isolated using fluorescent-activated cell sorter (FACS) based on their fluorescence intensity. However, because using the single colony as sample and cultivated to sort, the Endo-IL2 production of transformants sorting by FACS was not better than the original transformants. It still needed to continue study to solve this problem.