多肽_____]_多肽N乙酰氨基半乳糖转移酶2;

多肽:N乙酰氨基半乳糖转移酶2的原核表达、纯化及酶活检测

http://www.100md.com 《江苏大学学报(医学版)》 2006年第2期

多肽,,],多肽N乙酰氨基半乳糖转移酶2;,原核表达;,GST融合蛋白;,谷胱甘肽琼脂糖亲和层析;,酶活检测,1材料和方法,2结果

     [摘要] 目的：以人类多肽:N乙酰氨基半乳糖转移酶2(ppGalNAcT2)为研究对象,利用载体pGEX5X3在大肠杆菌(E. Coli)BL21中原核表达其编码序列,并对表达产物进行纯化、复性及酶活检测。方法：首先利用PCR技术从克隆载体pDONR201T2得到ppGalNAcT2全长编码序列,将其亚克隆至原核表达载体pGEX5X3,形成重组表达质粒pGEX5X3/T2。重组质粒转化大肠杆菌DH5α,测序鉴定后转化BL21,异丙基硫代半乳糖(IPTG)诱导后,表达产物为主要以包涵体形式存在的融合蛋白。对其复性后用谷胱甘肽琼脂糖(GlutathioneSepharose)4B亲和层析柱进行纯化。然后根据ppGalNAcT2的催化功能,设计底物,建立酶促反应体系,利用高效液相色谱(HPLC)进行酶活分析。结果：成功构建了原核表达重组载体pGEX5X3/T2, 通过蛋白质电泳检测到分子量约为90 kD的融合蛋白的表达,利用亲和层析得到纯化的酶蛋白,并经Western印迹得以验证。反应后体系上柱洗脱后出现酶促反应的产物洗脱峰。结论：成功构建了重组表达载体pGEX5X3/T2,ppGalNAcT2全长编码序列被成功表达、纯化及复性,检测到具有酶活性。

    [关键词] 多肽:N乙酰氨基半乳糖转移酶2; 原核表达; GST融合蛋白; 谷胱甘肽琼脂糖亲和层析; 酶活检测

    Prokaryotic Expression, Purification and Activity Assay of

    Human Polypeptide: Nacetylgalactosaminyltransferase2

    JIA Wei, HANG Saiyu, SHI Ning, ZHOU Yinghui, WU Shiliang

    (Department of Biochemistry and Molecular Biology, Medical School of Suzhou University, Suzhou Jiangsu 215123, China)

    [Abstract] Objective: To express the fulllength encoding sequence of human polypeptide: NAcetylgalactosaminyltransferase2 in Escherichia coli using vector pGEX5X3,and analyze its enzyme activity after purification and renaturation. Methods： The cDNA encoding ppGalNAcT2, which was amplified from plasmid pDONR201T2 by PCR, was subcloned into prokaryotic expression vector pGEX5X3, resulting in the recombinant plasmid pGEX5X3/T2 which was subsequently transformed into Escherichia coli BL21. A fusion protein was expressed after the induction by IPTG. After testified by western blot, we purified the expression products by GlutathioneSepharose affinity chromatography. After renaturation, its enzyme activity was assayed by Reverse Phase High Performance Liquid Chromatography (RPHPLC). Results： The plasmid pGEX5X3/T2 was reconstructed successfully. The fusion protein was expressed and testified by SDSPAGE and western blot. The purified enzyme was obtained by affinity chromatography and the HPLC showed its activity. Conclusion： The ppGalNAcT2 which has certain enzyme activity was successfully expressed, purified and renaturized. ......

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