虎鲸DHX36解旋酶表达纯化及酶学特性分析

Expression,Purification and Enzymatic Characteristics Analysis of Orcinus orca DHX36 Helicase

通过生物化学、生物信息学、生物物理学等方法系统地对虎鲸(Orcinus orca)DHX36解旋酶的酶学特性进行了分析。借助原核表达系统,表达纯化得到纯度大于95%的OrcDHX36蛋白。利用荧光各向异性法对OrcDHX36的底物结合活性进行研究,结果表明OrcDHX36可以特异性识别并结合G4DNA结构,但无法与ssDNA,dsDNA结合。通过停流光谱技术探索OrcDHX36解旋酶学特性,确定了其体外解旋的最适条件,明确了OrcDHX36的解旋方向为3′-5′。通过对比蛋白质信息数据库NCBI已公布的DHX36氨基酸序列,明确了大量DHX36蛋白(包括OrcDHX36在内)均无识别G4结构的RSM区;采用邻位法构建进化树,展示了OrcDHX36以及其他物种DHX36之间的亲缘关系。选择与OrcDHX36亲缘关系较远,且同样不具备RSM区的鸽子DHX36(ClDHX36),研究两者解旋活力特征,为揭示无RSM区的DHX36蛋白的G4解旋机制提供参考。

The enzymatic characteristics of Orcinus orca DHX36were systematically analyzed by biochemical,bioinformatics and biophysics methods.OrcDHX36 was successfully expressed and purified with a purity of more than 95%from the prokaryotic expression system.The substrate binding activities of OrcDHX36were determined by fluorescence anisotropy.These results indicate that OrcDHX36could specifically recognize and bind G4DNA structure,but could not bind ssDNA and dsDNA.The unwinding activities of OrcDHX36were studied by stopped-flow technology and optimized unwinding conditions for OrcDHX36was determined in vitro.The unwinding polarity of OrcDHX36was determined to be 3′-5′.Comparing with the protein information database on NCBI,it is clear that a large number of DHX36proteins(including OrcDHX36)have no RSM region to recognize G4structure.The phylogenetic tree was constructed by Neighbor-joining method to show the genetic relationship of OrcDHX36 and its orthologs in other species.The Columba DHX36(ClDHX36),which has a distant genetic relationship to OrcDHX36and also does not have the RSM region,was selected to compare with OrcDHX36in unwinding activity.This work may provide reference for clarifying the G4unwinding mechanism of DHX36without RSM region.