利用2A短胜肽連接不同綠色螢光蛋白質 探討螢光強度與目標蛋白質之間的關係

Abstract

Pichia pastoris 為嗜甲醇酵母菌，是常用的異源表達平台。藉由參數即時監測系統，可對醱酵過程作即時的判斷與調整，以避免汙染或是過多的原料浪費。然而目前尚無針對目標產物的即時監測工具，需經由取樣並進行特定的分析與定量才能確認其表現狀況。為使醱酵過程更有效率的生產目標蛋白質，本研究希望結合2A短胜肽與綠色螢光基因，將綠色螢光強度發展成醱酵製程中對目標蛋白質產量即時監控的參數。 根據實驗室早期研究的建構方式，以口足病毒 (foot and mouth disease virus) 可自我截切的2A短胜肽基因 (2A peptide gene)，連接目標蛋白質基因GMI-X與不同的綠色螢光蛋白質基因 (Green fluorescent protein, GFP)，其中包括常用的EGFP (Enhance green fluorescent protein, EGFP)，只會形成單體的mutated EGFP (EGFP A206K胺基酸突變, mEGFP)，及易降解的destabilized mEGFP (將mEGFP接上一段PEST sequence, dmEGFP)。利用胞內綠色螢光強度間接代表胞外目標蛋白質的表現量，以螢光流式細胞分選儀 (Fluorescent-activated cell sorter, FACS) 篩選高螢光強度的轉形株，並以搖瓶與醱酵槽進行甲醇誘導，分析不同綠色螢光蛋白質其螢光強度與目標蛋白質產量之間的關係。搖瓶誘導結果發現，原EGFP會因雙體化傾向，其螢光強度較形成單體的mEGFP弱，但兩者目標蛋白質產量與螢光強度變化具一致性。而dmEGFP因半衰期短，其螢光蛋白質不會過度累積於胞內，螢光強度較EGFP與mEGFP低，且無隨誘導時間增強之趨勢，但目標產物持續增加，其產量較EGFP與mEGFP的轉形株高。另外，醱酵槽結果也證實mEGFP與dmEGFP其綠色螢光強度可反映目標蛋白質表現量，且配合FACS分選得之轉形株皆為多拷貝數，因此未來即可根據不同的需求，選擇適當的建構方式作為目標蛋白質的即時監控系統。

The production of recombinant proteins plays an important role in the application of modern biotechnology. Microbial submerged fermentation is often used to produce the recombinant proteins using computer-controlled fermenter and appropriate induction strategy. The effective fermentation processes depend on real-time parameter monitoring, especially the production of target proteins. However, the production of the target proteins would not be detected until sampling and assay in most cases. Thus, it is important to have a real-time parameter to monitor the target protein production. In this study, a method for on-line monitoring of the target protein production in the methylotrophic yeast Pichia pastoris was developed by measuring the fluorescence intensity which was highly related to the heterologous gene expression. Three different polycistronic vectors were constructed. The vectors contain the AOX promoter, a signal peptide gene, the genes of GMI-X (an immunomodulatory protein from Ganoderma microsporum), a 2A peptides and EGFP, mutant EGFP (a point mutation A206K) or destabilized mEGFP, respectively. The high-yield transformats were sorted according to their fluorescence intensity using fluorescent-activated cell sorter (FACS). After methanol induction, the GMI-X extracellular supernatant was analyzed by ELISA and the intracellular EGFPs fluorescence intensity was measured by fluorescence spectrometry. The results showed that transformants of EGFP with the dimerization tendency fluoresced had less intensity than transformants of mutant EGFP, which tends to form monomer and soluble green fluorescent protein in cells. There was positive correlation between the fluorescence intensity and target gene expression in both EGFP and mEGFP transformants. Surprisingly, destabilized mEGFP was degraded quickly after translation due to the PEST sequence, and the fluorescence intensity of dmEGFP transformant remained at a low level, whereas the production of GMI-X still increased with the time of methanol induction. The highest yield of GMI-X was found in the dmEGFP transformant among three different EGFP forms. Also, the data from fermenter showed dmEGFP transformant had higher methanol utilization efficiency and GMI-X expression than mEGFP counterpart. Furthermore, all transformants sorted by FACS contained high copy numbers. In conclusion, the EGFP and mEGFP constructions provide a real-time parameter to monitor the target protein production, while the dmEGFP construction minimizes the EGFP burden and is feasible for elongated induction.