重组果蝇Merlin蛋白的表达与纯化  

Expression and Purification of Recombinant Drosophila Merlin

作　　者：张发友 周浩[1] Zhang Fayou;Zhou Hao(College of Life Sciences,Nankai University,Tianjin 300071,China)

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《南开大学学报（自然科学版）》2021年第1期35-40,47,共7页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(31870750,31670758)。

摘　　要：NF2基因的的缺失会导致Ⅱ型多发性神经纤维瘤的发生.NF2又称Merlin,和ERM蛋白家族成员类似,Merlin蛋白N端的FERM结构域与C端的CTD结构域存在分子内的相互作用,从而形成一种自抑制的闭合构象;当Merlin蛋白N、C端分子内的相互作用被破坏时,能释放出N端的FERM结构域,从而能与许多效应因子相互作用,形成一种伸展的打开的构象.果蝇Merlin蛋白的N、C端分子内的相互作用比人源Merlin更强,其构象的调节机制还不是很清楚.构建了原核表达质粒pET-28a-His_(6)-sumo-Merlin,通过大肠杆菌表达系统表达出了可溶性的重组果蝇Merlin蛋白,通过镍柱亲和层析,凝胶过滤层析获得了纯度较高、构象均一的重组蛋白Merlin.而且,还利用分析型超速离心分析了重组蛋白Merlin的构象,为进一步研究Merlin蛋白的三维结构及其构象的调节机制提供了一定的基础.The loss of NF2 gene can lead to the occurrence of type II multiple neurofibroma.NF2,also known as Merlin,like the ERM(Ezrin/Radxin/Moesin)family members,the intramolecular interaction between the N-terminal FERM domain and the C-terminal domian converts Merlin into an auto-inhibited closed conformation.When the intramolecular interaction between the N-terminal FERM domain and the C-terminal tail is destroyed,the FERM domain is released to bind with a variety of target effectors.The intramolecular interaction between the N-trminal FERM domain and the C-terminal domain in Drosophila Merlin is much stronger than in human Mer-lin,but how it is regulated is not well known.In the study,we constructed the plasmid of pET-28a-His-sumo-Merlin^(8-635),and expressed the recombinant protein His-sumo-Merlin^(^(8-635))..Soluble Merlin^(8-635)were purified through Ni-afnity chromatography,size exclusion chromatography.Besides,we analyzed the conformation of Merlin^(8-635)by ultracentrifugation.Conclusively,this research provided the basis for further research on the structure of Merlin^(8-635)and the regulation mechanism of the conversion of Drosophila Merlin’s conformation.

关 键 词：MERLIN 原核表达 层析 蛋白纯化 

分 类 号：Q51[生物学—生物化学]