大鼠PIAS3蛋白表达、纯化及生物信息学分析

Expression,purification and bioinformatics analysis of rat PIAS3

[目的]构建大鼠His-PIAS3真核表达载体,将His-PIAS3于体外进行表达纯化,并对PIAS3进行生物信息学分析。[方法]采用分子克隆构建His-PIAS3-pcDNA3.1真核表达载体,之后转染入HEK293细胞进行表达,考马斯亮蓝染色检测整体蛋白变化,免疫印迹鉴定His-PIAS3蛋白表达及细胞整体蛋白SUMO化,镍柱纯化His-PIAS3蛋白。生物信息学方法分析大鼠PIAS3蛋白的理化性质、亲疏水性、二级结构及序列的保守性。[结果]His-PIAS3目的基因扩增成功,且亚克隆入pcDNA3.1载体;和空载体组相比,转染His-PIAS3-pcDNA3.1组PIAS3蛋白表达水平明显增加,且整体蛋白的SUMO化水平显著升高;镍柱纯化可获得较高纯度的His-PIAS3蛋白。生物信息学结果显示,大鼠PIAS3蛋白包含628个氨基酸残基,分子量为68.3 kDa,理论等电点为8.05,亲水性平均值为-0.242;PIAS3主要定位于膜内;PIAS3蛋白的二级结构主要由18.79%α-螺旋、13.69%延伸链和67.52%无规卷曲组成;不同物种间pias3序列相似性均在70%以上。[结论]His-PIAS3真核表达载体构建成功;His-PIAS3可在HEK293细胞中高效表达,具有较高酶活性;获得较高纯度His-PIAS3蛋白;PIAS3主要定位于核内,为碱性、亲水性不稳定蛋白,具有高度保守性。

[Objective]Rat His-PIAS3 eukaryotic expression vector was constructed,and then His-PIAS3 was expressed and purified in vitro,followed by bioinformatics analysis.[Method]The His-PIAS3-pcDNA3.1 eukaryotic expression vector was constructed by molecular cloning,and then transfected into HEK293 cells for expression.Coomassie brilliant blue staining was sent to detect the changes of overall protein.Immunoblotting was used to detect the expression of His-PIAS3 protein and SUMOylation in cells.Ni-NTA was performed to purify His-PIAS3 protein.Bioinformatics methods were used to analyze the physicochemical properties,hydrophobicity,secondary structure and sequence conservation of rat PIAS3 protein.[Result]The gene of His-pias3 was amplified successfully and subcloned into pcDNA3.1 vector.Compared with the empty vector transfected group,the levels of PIAS3 protein and whole protein SUMOylation were significantly increased in the His-PIAS3-pcDNA3.1 transfected group.Moreover,His-PIAS3 protein with high purity was obtained by Ni-NTA purification.In addition,the bioinformatics results showed that the rat PIAS3 protein contained 628 AAs,and the molecular weight was about 68.3 kDa.Theoretical isoelectrical point was 8.05,and the average hydrophilicity was-0.242.The secondary structure of PIAS3 protein was mainly composed of 18.79%α-helix,13.69%extended chain,and 67.52%random coil.The similarity of pias3 sequences among different species was more than 70%.[Conclusion]The His-PIAS3 eukaryotic expression vector was successfully constructed.His-PIAS3 was expressed effectively in HEK293 cells,and had high enzymatic activity.High purity His-PIAS3 protein was obtained.PIAS3 is mainly localized in the nucleus,and is a basic hydrophilic unstable protein with high conservation.