牛痘病毒拓扑异构酶Ⅰ的原核表达与纯化

Prokaryotic expression and purification of topoisomeraseⅠ of vaccinia virus

【目的】建立牛痘病毒拓扑异构酶Ⅰ(Vaccinia topoisomeraseⅠ)的原核表达体系及纯化方法,以获得高活性、高纯度的牛痘病毒拓扑异构酶Ⅰ。【方法】优化并人工合成牛痘病毒拓扑异构酶Ⅰ的全基因序列,构建牛痘病毒拓扑异构酶Ⅰ的原核表达载体(pET28a/TOPO)并转化E.coliBL21 Star(DE3)菌株,优化其诱导表达条件,表达蛋白经Talon his-tag purification resin螯合层析柱纯化,用SDS-PAGE鉴定纯化效果,借助酶切质粒pLLP-OmpA检测纯化蛋白的活性。【结果】转化pET28a/TOPO的E.coliBL21 Star(DE3)特异地表达了牛痘病毒拓扑异构酶Ⅰ蛋白,最佳诱导条件为30℃下以0.5 mmol/L IPTG诱导12 h。该蛋白经钴离子螯合层析柱纯化后,SDS-PAGE电泳结果显示其为分子质量36 ku的蛋白。酶切和冻融试验结果表明,该酶具有高活性和高稳定性。【结论】成功构建了牛痘病毒拓扑异构酶Ⅰ的原核表达和纯化体系,为后续拓扑异构酶Ⅰ的应用研究奠定了基础。

【Objective】 The study was done to obtain high-purity and high-activity topoisomeraseⅠfrom vaccinia virus by establishing the prokaryotic expressing and purification system.【Method】 The sequence encoding Topoisomerase Ⅰ optimized by the usage of the favourite codons in E.coli was synthesized and inserted to expression plasmid pET28a（＋）.The expression topoisomerase Ⅰ was induced in E.coli BL21 Star（DE3） by IPTG.The protein was purified by Talon his-tag purification resin and characterised by SDS-PAGE to examine the purified protein.The activity of topoisomeraseⅠwas assayed by digestion of plasmid pLLP-OmpA as the substrate.【Result】 The optimum conditions of expression were 30 ℃,0.5 mmol/L IPTG,12 h.High yield and high purity of topoisomeraseⅠwas achieved by Co2＋ affinity chromatography,the purified protein was assayed by SDS-PAGE and the result was one single band（with molecular weight of 36 ku）.Activity analysis and freeze-thaw resistance analysis showed that the recombinant topoisomerase had high activity and high stability.【Conclusion】 The prokaryotic expressing system and purification method were constructed and optimized successfully.These methods can be used in the further study of topoisomerase Ⅰ.