厚壳贻贝足丝超氧化物歧化酶的重组表达及功能

Recombinant Expression and Functional Analysis of Mussel Byssal Superoxide Dismutase

为探明厚壳贻贝足丝中一种新型超氧化物歧化酶(Byssal superoxide dismutase,BSOD)类蛋白的结构与功能,对厚壳贻贝BSOD开展了序列分析并采用密码子优化结合大肠杆菌重组表达系统,开展BSOD重组表达以及表达产物的分离纯化和功能鉴定。序列分析结果表明,厚壳贻贝BSOD序列中含信号肽和典型的SOD结构域,属于分泌型SOD分子;其一级结构与数据库中已报道的其他物种SOD具有一定的相似性,但进化树分析结果表明厚壳贻贝BSOD是一种较为新颖的SOD分子,与水生甲壳类生物的胞外SOD同源。重组表达结果表明,重组BSOD主要通过包涵体形式表达。经复性后,重组厚壳贻贝BSOD具有明显的歧化酶酶活性;该活性在重组BSOD鳌合铜离子和锌离子后明显增强;且鳌合了铜离子和锌离子后重组BSOD对DPPH具有较强的清除率,表明BSOD是一种需要金属离子辅助的抗氧化蛋白。推测BSOD在足丝黏附过程中参与了其他富含多吧的足丝蛋白的保护以及还原态维持,并对贻贝足丝在固化和黏附前维持还原态具有重要意义。

For understanding the structure and function of a novel byssal superoxide dismutase(BSOD)of Mytilus coruscus,sequential analysis and recombinant expression with codon optimization of BSOD were performed.BSOD has a signal peptide and a SOD domain in its sequence,and the homology searching results together with cladogram showed that BSOD is a novel byssal SOD with sequential similarity with the SOD molecules from aquatic crustaceans.The recombinant BSOD was expressed mainly in inclusion body of Escherichia coli.After purification and renaturation,the recombinant BSOD showed high enzymatic activity,as well as high DPPH radical scavenging activity.This activity significantly increased after copper and zinc ions chelating,indicating that the antioxidant activity of recombinant BSOD is associated with the divalent metal ion.The results also provided the foundation for further understanding of the roles of BSOD in adhesion and solidification of Mytilus byssus.In addition,recombinant BSOD has the potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage.