Periaxin蛋白中PDZ结构域的重组表达与纯化  

Recombinant Expression and Purification of Periaxin-PDZ Domain

作　　者：石亚伟[1] 任燕[1] 刘俊林[1] 杨廷[1] 

机构地区：[1]教育部化学生物学与分子工程重点实验室,山西大学生物技术研究所,山西太原030006

出　　处：《山西大学学报（自然科学版）》2012年第1期118-121,共4页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(31170748);生物大分子国家重点实验室2010年开放课题

摘　　要：Periaxin属于有髓施旺细胞非致密髓鞘中的重要蛋白之一,该蛋白的突变,会引起腓骨肌萎缩症4F亚型发生.文章以小鼠periaxin基因为模板,PCR扩增编码Periaxin-PDZ及其延长型的基因序列,插入pETM-3C表达载体中.转入E.coli BL21(DE3)中,经IPTG诱导表达重组蛋白.再经Ni 2+-NTA亲和柱及Sephacryl S-200凝胶层析获得电泳纯的Periaxin-PDZ蛋白.荧光光谱法分析Periaxin-PDZ和脂膜的结合能力,结果显示Periaxin-PDZ(18-102aa)与脂质体的解离常数为Kd=4.415μg/mL,延长型Periaxin-PDZ(18-129aa)与脂质体的解离常数为Kd=7.265μg/mL,Periaxin-PDZ(18-102aa)与脂膜的结合能力略高于延长型Periaxin-PDZ(18-129aa).Periaxin is a protein of noncompact myelin in the peripheral nervous system.The loss-of-function mutation of the gene encoding periaxin can cause demyelinating Charcot-Marie-Tooth 4F disease.The gene of periaxin-PDZ was amplified by PCR with pBluescrip+SK(Ⅱ)-periaxin as the template and inserted into the vector pETM-3C to form a recombinant plasmid pETM-3C-pdz.The recombinant protein was overexpressed in Escherichia coli BL21(DE3) by IPTG induction.A purification procedure for periaxin-PDZ was performed by Ni2+-NTA column and Sephacryl S-200 column.The lipid membrane binging capacity of the Periaxin PDZ domain was evaluated with fluorescence spectroscopy.The results show that dissociation constants of lipid membranes with Periaxin-PDZ(18-102aa)or Periaxin-PDZ(18-129aa)is 4.415 μg/mL or 7.265 μg/mL respectively.The lipid membrane binging capacity of Periaxin-PDZ(18-102aa)is slightly higher than the extended Periaxin-PDZ(18-129aa).

关 键 词：腓骨肌萎缩症 Periaxin蛋白 pdz结构城 脂质体 荧光光谱法 

分 类 号：Q816[生物学—生物工程]