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

目的：以人类多肽：N-乙酰氨基半乳糖转移酶2（ppGalNAc-12）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伞长编码序列被成功表达、纯化及复性，检测到具有酶活性。

Prokaryotic Expression, Purification and Activity Assay of Human Polypeptide: N-acetylgalactosaminyltransferase2

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.