II.在之前學長的研究重組酵母菌醛類去氫酶 ALD6p 並轉型至大腸桿菌( Escherichia coli )的表現載體 BL21，以達到大量誘導表現重組蛋白之目的。但由於之前一直存在目標蛋白質降解之問題，故我們透過降低培養時的溫度以改善目標蛋白質降解問題。另外也利用陰離子交換樹脂Q SepharoseTM Fast Flow ( Amersham Biosciences )採用反向吸入欲純化之樣品，正向洗出雜蛋白質並搭配目標蛋白質pI之適當 pH 值的 Buffer 以達到一步純化( one-step purification )之目的。 I.The ORF of YJR129Cp of S. cervisiae has a close match with the methyltransferase in database, therefore, in this research the putative methyltransferase YJR129Cp is used as enzyme to find the substrate of YJR129Cp of S. cervisiae by means of the substrate of ΔYJR129Cp of cell lysate as well as 2-D electrophoresis procedure.
Thus far, the translation factor 2 (EF-2) has been identified as a methylation substrate forYJR129Cp in S. cervisiae by proteomics analysis. Afterward, EF-2 will be purified to make sure the substrate of putative methyltransferase YJR129Cp. Also, after it is confirmed we want to analyze further that methyltransferase YJR129Cp methylates substrate in a way with the physiological significance.
On the other hand, this research is proceeded with isoelectric focusing (IEF) and SDS-PAGE in a separate manner. For this reason, we are able to congregate a large quantity of focus bands after IEF is analyzed. And we can extract protein from some of them and then concentrate the protein to carry out SDS-PAGE. In this way, even though they are only lightly exposed, the exposed focus bands are still enough by combination of multiple bands. Therefore, we can gather a plenty of protein samples to benefit proteomics analysis.
II.In the previous research the recombinant ALD6p of S. cervisiae has been overexpressed in E. coli strain. However, the degradation problem persisted. We solved this problem by way of decreasing the temperature of induction. Moreover, we used Q SepharoseTM Fast Flow ( Amersham Biosciences ) and reversed the flow direction of elution relative to that of sample loading, and also referred to ALD6p pI value to select for suitable buffer pH to fulfill the purpose of one-step purification.