李冠群Guan-Chiun Lee邱家鴻Chia-Hung Chiu2019-09-052014-8-272019-09-052013http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060043048S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104056嗜甲醇酵母菌 (Pichia pastoris) 系統已經被廣泛使用在重組蛋白的表達。此系統具有真核生物的優點，例如轉譯後修飾、蛋白可分泌至胞外、生長快速、以及其內毒素與病毒汙染的風險較低。Glyceraldehyde-3-phosphate dehydrogenase (GAP) 的啟動子 (PGAP) 在 P. pastoris 系統中常被使用當作重組蛋白持續性表達的啟動子。這個持續性表達的 PGAP 系統具有穩定的基因表現特性，且因表達基因時無需使用誘導物，不會造成不同細胞間的轉錄程度異質性。經基因工程改造過後所得到的不同強度啟動子，可以用來調控基因表現以得到適當的蛋白表達量。為了增進在 P. pastoris 系統中蛋白表達量，本研究透過針對 GAP 啟動子序列隨機突變，建立一個突變庫以便篩選得到強的啟動子。篩選方法是使用抗抗生素 zeocin 基因 (即Streptoalloteichus hindustanus bleomycin gene, Sh ble gene) 當作報導基因。我們使用基因置換的轉型方式以去除多套插入的發生，避免套數多寡造成對啟動子篩選上的干擾。在高濃度的 zeocin 中，我們從 708 支轉型株中篩選到三支存活的突變株。經基因定序確認可能會影響啟動子強弱的突變位置。這些具較強轉錄活性的突變啟動子可以被應用在提高其他重組蛋白的表達。The methylotrophic yeast Pichia pastoris is a well-established protein production host. It has the advantages of eukaryotes, such as eukaryotic post-translational modifications, efficient protein secretion, fast growth on economical media, and little risk of contamination with endotoxin or virus. The glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (PGAP) has been used for constitutive expression of heterologous proteins. The constitutive PGAP system permits steady-state gene expression and prevent the transcriptional heterogeneity in inducible expression systems. Engineered promoters with various strengths are useful genetic tools that enable the precise control of gene expression to obtain optimal yield. To improve the production of recombinant protein in P. pastoris, we created a PGAP library through random mutagenesis. Antibiotics Zeocin resistant gene (Streptoalloteichus hindustanus bleomycin gene, Sh ble gene) was used as a reporter. We performed transformation through gene replacement to rule out the multi-copy insertion events which may interfere with the promoter selection. 3 survival mutants were selected from 708 transformants under higher Zeocin concentrations. We identified the important mutation sites in the GAP promoter regions that may cause the variant strengths. These strong mutant promoters could be used to improve the expression of other recombinant proteins.嗜甲醇酵母菌啟動子隨機突變篩選Pichia pastorispromoterrandom mutagenesisselection