應用THUB標籤在蛋白質的表達與純化上

Application of Tat-derived peptide-His6-Ubiquitin-HLA-B27 peptide (THUB) tag in protein expression and purification

游智翔(Chih Hsiang Yu)

指導教授 ： 黃憲斌

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2014年)

摘要

Tat胜肽His6-Ubiquitin-人類白血球抗原-B27胜肽(THUB)被我們實驗用來運送抗原性胜肽到內質網(ER)的內腔。在以前的研究中我們發現 THUB與鎳離子樹酯、SP樹酯有很強的結合力，讓這個融合蛋白可用在蛋白質表達與純化時很好的標籤。要表達的蛋白質可接在類白血球抗原-B27胜肽的COOH端，表達的蛋白質可以利用兩步驟層析法從初萃取液中純化出。再利用酵母菌ubiquitin C端水解酶(YUH-1)把表達的蛋白質切出、而釋放的表達蛋白可利用鎳離子樹酯層析純化出。本研究，我要來檢驗這個假設。脂蛋白元E3(1-191) (ApoE3(1-191))接在THUB的COOH端，THUB-ApoE3(1-191)在大腸菌BL21(DE3)中表達，利用兩步驟層析法純化出THUB-ApoE3(1-191)。然而，YUH-1沒辦法有效切出B-ApoE3(1-191).

關鍵字

標籤

並列摘要

The Tat-derived peptide-His6-ubiquitin-HLA-B27 peptide (THUB) has been used in our laboratory for delivery of the antigenic peptide to the lumen of the endoplasmic reticulum (ER). In the previous studies, we have found that THUB strongly binds to both Ni2+-Sepharose and SP-Sepharose, allowing this fusion protein to be able to serve as a good tag for protein expression and purification. The expressed protein can link to the C-terminal of HLA-B27 peptide. The expressed protein can be easily purified from the crude extract by a two-step chromatography. The expressed protein can be cut out from the fusion protein by yeast ubiquitin C-terminal hydrolase and the released protein can be easily separated from THU by the Ni2+-Sepharose chromatography. In this study, I tested our hypothesis. Apolipoprotein E3 (ApoE3) was linked to THUB and the THUB-ApoE3(1-191) was expressed in E. coli BL21(DE3). THUB-ApoE3(1-191) can be purified by a two-step chromatography. However, we found that YUH-1 cannot efficiently cleave the THUB-ApoE3 into THU and B-ApoE3(1-191).

並列關鍵字

THUB

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