OGG1与Smad7蛋白相互作用的具体区域鉴定

Identification of specific regions of interaction between OGG1 and Smad7 protein

目的体外探究8-氧鸟嘌呤DNA糖化酶1(OGG1)蛋白和Smad家族成员7(Smad7)蛋白相互作用的具体区域。方法利用原核表达系统表达OGG1和Smad7的蛋白截段体,通过GST蛋白纯化和pull-down实验验证两个蛋白不同截段体间的相互作用。结果将OGG1与Smad7进行pull-down实验,发现两者存在相互作用。将含有1-346个氨基酸(1-346 aa)全长片段的OGG1及其截段体1-242 aa、1-138 aa、1-63 aa和34-346 aa,与Smad7全长(1-428 aa)进行pull-down实验,发现OGG1全长(1-346 aa)及其截段域(34-346 aa)与Smad7具有相互作用。将OGG1截段域(34-346 aa)与Smad7全长(1-428 aa)及其截段体1-308 aa、1-240 aa和1-128 aa进行pull-down实验,发现只有Smad7截段体1-128 aa与OGG1截段体34-346 aa不存在相互作用。结论初步判断OGG1(34-346 aa)和Smad7(128-240 aa)可能为OGG1与Smad7发生相互作用的区域。

Objective To explore the specific region of interaction between 8-oxyguanine DNA glycosylase(OGG1)and Smad family member 7(Smad7)proteins in vitro.Methods The protein truncates of OGG1 and Smad7 were expressed using prokaryotic expression system,and the interactions between the different truncates of the two proteins were examined by GST protein purification and pull-down experiments.Results OGG1 full-length(1-346 aa)containing a fragment of amino acids 1-346(1-346 aa)and its truncators 1-242 aa,1-138 aa,1-63 aa and 34-346 aa were subjected to pull-down experiments with full-length Smad7 protein.The full length(1-346 aa)and truncated fragment(34-346 aa)of OGG1 were found to interact with full-length Smad7 protein.Pull-down experiment was also used to investigate the interactions between truncated fragment of OGG1(34-346 aa)and full-length fragment of Smad7(1-428 aa)and its truncators 1-308 aa,1-240 aa and 1-128 aa,and the result showed that only the truncated Smad7(1-128 aa)did not interact with the truncated OGG1(34-346 aa).Conclusion It is preliminarily determined that OGG1(34-346 aa)and Smad7(128-240 aa)might be the interaction region between OGG1 and Smad7.