鸡SUMO1的HIS/GST双融合纯化标签载体的构建及表达分析

Construction and characterization of expression vector harboring chicken SUMO1 fused by His/GST ditags

SUMO(small ubiquitin-related modifier,小泛素样相关修饰物)是一种重要的蛋白翻译后修饰物,对调节蛋白质的活性、功能和细胞定位都起着至关重要的作用。虽然SUMO和泛素的空间结构很相似,但是发挥的功能却有很大的不同,SUMO不像泛素那样介导蛋白质的降解,反而可以增强蛋白质的稳定性。近年来,研究发现人的SUMO作为一种融合标签和分子伴侣来可以显著增加一些难溶解蛋白的可溶性表达。本研究以原核表达载体pET-28a为基础构建了鸡SUMO1重组质粒HIS-SUMO1-GST-pET28a,并在SUMO1和GST之间插入IL2得到重组质粒HIS-SUMO1-IL2-GST-pET28a,经IPTG诱导表达,并分别使用Ni-NTA及Glutathione-sepharose 2次亲和层析纯化,获得了纯度较高的融合蛋白。活性分析发现去SUMO化酶人的SENP1可完全切开纯化融合的蛋白经。结果表明,此SUMO融合的HIS/GST双标签表达载体不但可以用于提高难溶蛋白的溶解度,而且表达产物也作为去SUMO化酶研究的底物。

As a post-translational protein modifier,SUMOs（small ubiquitin-related modifier）play important roles in regulating protein activity,function and cellular localization.Although SUMOs are structurally similar to ubiquitin,their functions are distinct.Ubiquitin can lead to degradation of attached proteins,but SUMOs can improve stability and folding of the target protein.In present study,we constructed the recombinant plasmid by inserting HIS/GST ditags and chicken SUMO1 based on pET-28 avector backbone.Chicken IL2 was cloned into this recombinant vector to test the improvement in solubility and expression level of the recombinant protein HIS-SUMO1-IL2-GST.The fusion protein was purified with ditags.His-SUMO1 could be effectively removed by SENP1.The results suggested chicken SUMO1 with HIS/GST ditags could improve the solubility and expression level of some difficult-to-express proteins as well as to be used as a substrate in SUMO research.