双分子荧光互补系统分析S-periaxin蛋白的聚合

S-periaxin蛋白是施旺氏细胞特异性表达的一种蛋白,在维持髓鞘的稳定方面发挥重要作用,该蛋白基因的突变引起腓骨肌萎缩症4F亚型的发生。Periaxin基因由于mRNA剪切方式的不同可以编码两种长短不同的含PDZ结构域的蛋白,即L-periaxin和S-periaxin。两种蛋白在施旺氏细胞的定位存在明显的差异,相对L-periaxin而言,S-periaxin无论是分子结构还是生物学功能均未见相关研究。该文从大鼠的施旺氏细胞系RSC96克隆了S-periaxin基因,构建了原核表达载体pETM-3C-S-periaxin,在大肠杆菌中进行重组表达,经Ni-NTA亲和柱和Sephacryl S-200凝胶层析柱获得电泳纯的目的蛋白。体外戊二醛交联分析蛋白的聚合状态表明,S-periaxin蛋白在体外易于形成不同聚合度的聚合物。免疫共沉淀也表明,S-periaxin蛋白存在同源蛋白间相互作用。另外,构建了原、真核双分子荧光互补系统,并利用该系统分析了细胞内S-periaxin蛋白间的相互作用。

Analysis of Protein Interaction Between S-periaxin by Bimolecular Fluorescence Complementation Assay

Periaxin is expressed by myelinating Schwann cells and plays an essential role in stabilizing the Schwann cell-axon unit in the myelinated fibers of the vertebra. Mutations in the periaxin gene are known to cause autosomal recessive demyelinating Charcot-Marie-Tooth （CMT4F）. Periaxin gene encodes L-periaxin and a truncated isoform, S-periaxin, which have an N-terminal PDZ domain and are targeted differently in the Schwann cell. The molecular structure and biological function of S-periaxin are unknown to date. In this work, the DNA sequence encoding S-periaxin was cloned into the vector pET-M-3C to form a recombinant plasmid pET-M-3C-S-periaxin. The recombinant protein was overexpressed in E.coli BL21 and purified by Ni-NTA column and Sephacryl S-200 column. S-periaxin was easy to form the different degree of polymerization in vitro by glutaraldehyde crosslinking. S-periaxin could homodimerize with coimmunoprecipitation. In addition, bimolecular fluorescence complementa- tion （BiFC） system was developed by splitting mCherry. BiFC demonstrated S-periaxin could also form homodimer in E.coli BL21 and RSC96 cell.