锌指蛋白ZBTB20原核重组表达与纯化体系的优化

Optimization for the Recombinant Expression and Purification of Zinc Finger Protein ZBTB20

目的优化锌指蛋白ZBTB20的谷胱甘肽巯基转移酶(GST)融合蛋白表达体系和纯化条件,以获得足够的ZBTB20蛋白及其截短体的GST融合蛋白,为研究该蛋白的转录调控机制奠定基础。方法将ZBTB20蛋白及其截短体的GST融合蛋白表达质粒转化大肠杆菌BL21并进行异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。通过SDS-PAGE电泳和考马斯亮蓝染色鉴定融合蛋白的表达量;对诱导表达的温度与时间、包涵体裂解液的配方和裂解方法以及纯化方法进行优化。结果经SDS-PAGE和考马斯亮蓝染色发现在37℃诱导条件下全长ZBTB20-GST蛋白主要以包涵体形式表达且不易溶于包涵体裂解液中。采用低温诱导可以提高包涵体中全长ZBTB20-GST融合蛋白的溶解性。改良的纯化方法能够将包涵体裂解上清中的全长ZBTB20-GST融合蛋白纯化下来。ZBTB20蛋白截短体的溶解性比全长蛋白好,且截短体越短溶解性越好。结论优化后的融合蛋白诱导表达和纯化方法能够获得足够的ZBTB20蛋白及其截短体的GST融合蛋白用于后续研究。

Objective To optimize the expression and purification conditions of glutathione S-transferase(GST)fusion protein of the zinc finger protein ZBTB20.Methods GST-tagged ZBTB20 plasmid and its truncates were transformed into the host strain BL21.The expression of fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside(IPTG).The fusion protein was analyzed by SDS-PAGE and Coomassie brilliant blue staining.Some critical experimental parameters were optimized,which include expression temperature,induction time,inclusion body solubilization,and protein purification.Results The GST-tagged ZBTB20 was mainly expressed as inclusion bodies and remained insoluble in lysis buffer.Lowering induction temperature to 20℃improved the solubility of GST-tagged ZBTB20.By using optimized binding buffer with appropriate detergents,GST-tagged ZBTB20 protein was successfully purified by affinity chromatography.The solubility of ZBTB20 truncates was much better than that of its full-length protein.Conclusion Sufficient GST-tagged ZBTB20 protein and its truncates were obtained by using the optimized expression and purification protocols,which will be applicable to identify the potential interacting molecules of ZBTB20.