| Summary: | 碩士 === 淡江大學 === 化學學系碩士班 === 101 === 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.
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