大鼠冠蛋白6在大肠杆菌中的表达、纯化与复性

Expression,purification and renaturation of rat Coronin 6 in Escherichia coli

目的利用大肠杆菌表达体系制备冠蛋白6(Coronin 6),探讨其表达、纯化和复性的条件与方法。方法构建Coronin 6基因过表达载体,转化大肠杆菌BL21(DE3)进行诱导表达,对纯化得到的蛋白质进行复性,利用镍亲和层析柱和分子筛层析柱进行纯化,SDS-PAGE检测蛋白质表达和复性情况。结果成功构建Coronin 6过表达载体;Coronin 6在大肠杆菌BL21(DE3)中能够大量表达,表达条件为37℃、异丙基-β-D-硫代半乳糖苷0.1mmol/L诱导6h,Coronin 6以包涵体的形成存在。SDS-PAGE分析表明,Coronin 6蛋白质能够充分溶解于8mol/L的尿素中,其分子量为52.9kDa。对Coronin 6进行纯化和复性,获得较高纯度(>90%)的Coronin 6活性蛋白质,经纯化后能稳定存在于凝胶层析缓冲液中,每升LB培养基可获得纯度较高的蛋白质2.1mg。结论获得纯度较高的Coronin 6活性蛋白质,克隆并建立Coronin 6蛋白在大肠杆菌中表达、纯化和复性的条件。

Objective To investigate the condition and methods for the expression,purification and renaturation of rat Coronin 6 in Escherichia coli.Methods The overexpression vector of Coronin6 was established and transfered into Escherichia coli.The protein was obtained,renatured and purified.The expression and renaturation of protein were detected by SDS-PAGE.Results The overexpression vector of Coronin 6 was constructed successfully.Coronin 6 expressed as inclusion body in Escherichia coli in the condition of 37℃ and isopropyl-beta-D-thiogalactoside 0.1 mmol/L for6 hours.The protein of Coronin 6 dissolved completely in urea 8 mol/L and the molecular weight was52.9 kDa.After purification and renaturation,the high purity protein of Coronin 6 was obtained,which existed stably in gel chromatography buffer.The high purity protein 2.1 mg was obtained in 1 LLB medium.Conclusion High purity active protein of Coronin 6 is obtained and the conditions for the expression,renaturation and purification are established.