第I部分： 應用高效率電穿孔法於酵母菌的雙雜合篩選， JHD1/YER051W為啤酒酵母菌(Saccharomyces cerevisiae) BY4742 上的一段ORF，為去甲基酶；將JHD1 ORF接上BD載體，作為【餌】，由已接上 AD載體之酵母基因的基因庫，作為【魚】。送入啤酒酵母菌 PJ69-4A中，會彼此相互結合的蛋白質就能在篩選的特定培養基中表現出來。再把篩選出的基因作進一步分析，可以瞭解到該蛋白之功能與特性。目前在JHD1/YER051W 的酵母菌雙雜合篩選實驗中，已篩選出的基因為CIN5/YOR028C。 第II部分： Bacillus subtilis TKU007中含有的納豆激酶基因，前面另有一段含有77個胺基酸的propeptide，為蛋白原轉變成活性蛋白質過程中去除的肽段。本研究利用PCR (聚合酶鏈反應)大量生產含有propeptide的納豆激酶基因片段( pronattokinase )與納豆激酶基因片段(nattokinase)，並在兩者的C ’端接上His•Tag coding sequence後，轉型到pYES2表現載體上進行異源表現，使用半乳糖去誘導產生蛋白質，及其蛋白質表現後的純化。 In part I, high efficiency transformation by electroporation is applied to yeast two¬- hybrid screening. JHD1/YER051W is a demethylase gene from Saccharomyces cerevisiae BY4742. The JHD1/YER051W ORF is ligated to the BD vector as the “bait”. Appropriate DNA library constructed on the AD vector was screened for “fish” when the library was transformed into yeast PJ69-4A. The interacting proteins expressed from the AD vector would be selected in the specific medium. Afterwards, more functional analysis will be done to further understand the functional role of the interacting proteins. In this study, JHD1 was found to interact with CIN5/YOR028C gene product using the yeast two-hybrid screening. In part II, a nattokinase from Bacillus subtilis TKU007 was investigated. Propeptide, which is 77 amino acids fragment at the N terminus of the nattokinase, is required to convert pronattokinase to the mature active nattokinase. The propeptide is then cleaved off by the mature nattokinase. In this research, we used PCR (polymerase chain reaction) to clone pronattokinase and nattokinase sequence, respectively. Both of them are linked to His•Tag coding sequence at their C termini, and ligated to the expression vector pYES2. Heterologous expression in yeast was induced by galactose, and the over-expressed protein was purified .
