第一部分研究啤酒酵母菌中ALD4p對粒線體型態所造成之影響，我們分別將建構之ALD4-GFP-pYes2和不含ALD4基因序列之pYes2轉形至酵母菌BJ2168，在誘導其蛋白質表現並利用Mito-Tracker Red FM將粒線體染色後，透過螢光顯微鏡觀察過度表現ALD4p對粒線體型態的影響，結果發現當過度表現ALD4p時，顆粒狀的粒線體比例確實明顯上升，管柱狀的粒線體比例明顯下降，藉由流式細胞儀確認ALD4-GFP蛋白確實大量表現，接著將點突變後的ALD4C324S-GFP-pYes2轉形至酵母菌BJ2168，誘導其蛋白質表現並利用Mito-Tracker Red FM將粒線體染色後，藉由螢光顯微鏡觀察失去活性的ALD4p對粒線體型態之影響。 第二部份我們將啤酒酵母菌中之TEF1基因與pGEX-4T-1酶接後轉形至大腸桿菌BL21中，並表現TEF1-GST融合蛋白，再利用GST純化所需之重組蛋白，而在SDS膠片分析時，TEF1-GST融合蛋白無法大量表現於上清液中，GST純化也無法將其有效地單獨分離出來，而仍含有其它分子量的蛋白質，可能會影響後續之研究，因此我們將已經建構好之TEF1-GST基因再接上帶有His-tag融合蛋白基因之載體pET28c，希望在利用GST純化前，先藉由His-tag純化將欲得到之TEF1-GST-His蛋白與其他可能會影響後續研究之蛋白分離後，再使用GST純化，以提高其純度，並且將表現系統換成酵母菌以使融合蛋白大量表現於上清液。 In part I, we transformed yeast strain BJ2168 with ALD4-GFP-pYes2 and pYes2, and induced protein expression with galactose. We stained yeast mitochondrial by Mito-Tracker Red FM and observed the morphological change under fluorescence microscopy. We found that mitochondria and ALD4-GFP fluorescence overlap partially, indicating that mitochondrial morphological changes may be due to activity of ALD4p. Then we used flow cytometery to analyzed the ALD4-GFP protein quantitatively, and confirmed the expression of ALD4-GFP protein. We also inactivated ALD4p catalytic activity to verify it being the activity rather than the overexpression protein which effects morphological change of mitochondria. In part II, we expressed TEF1-GST fusion protein after constructing TEF1-pGEX-4T-1 into E.coli. TEF1-GST fusion protein could not expressed in supernatant numerous, and still had other protein after we used GST column to purified, and those proteins might affect subsequent research. So we constructed and expressed TEF1-GST-pET28c and used His-tag column to purified before used GST column to avoided those proteins. We wanna constructing TEF1-GST-His-pYES2 into yeast to change expression system, and improve about fusion protein expression in supernatant.
