浙江枝吻纽虫肌动蛋白基因的原核表达及其重组蛋白的体外自组装  

Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro

作　　者：李晔[1] 陈蕾[1] 周君[1] 李成华[1] 苏秀榕[1] 李太武[2] 

机构地区：[1]宁波大学海洋学院,宁波315211 [2]宁波城市职业技术学院,宁波315100

出　　处：《生物技术通报》2013年第8期113-118,共6页Biotechnology Bulletin

基　　金：浙江省自然基金资助项目(Y3100480);浙江省教育厅科研项目(20070951)

摘　　要：肌动蛋白(actin)是细胞骨架的主要成分。细胞内肌动蛋白通过与actin结合蛋白(actin binding proteins,ABPs)相互作用,形成以F-actin为基础多种ABPs参与装配的高度有序的超分子聚合结构,行使各种重要生理功能。根据已获得的浙江枝吻纽虫actin的全长cDNA序列,构建actin的原核表达质粒pET-28a-A,并转化至E.coliBL21菌株,经IPTG诱导后,重组蛋白以包涵体的形式被大量表达。重组蛋白经Ni-NTA Agarose亲和层析分离纯化后,进一步透析复性,SDS-PAGE显示纯化复性后的r-actin的相对分子量约为43 kD,在体外聚合条件下,采用激光原子力显微镜(atomic forcem icroscope,AFM)技术,对r-actin通过自装配过程形成的大尺度聚集结构进行观察和分析。研究发现,r-actin在体外通过自装配过程除了形成无序的蛋白堆积物之外,还能够聚合形成复杂的离散结构,包括树状分支的纤维丛、无规卷曲的纤维簇等。Actin自装配过程反映了其固有的聚合热力学特性,深入探索将有助于进一步研究ABPs在体内actin超分子聚合结构体系装配中的调控作用及其分子机制。Actin is a major component of cytoskeleton.In cells,actin associates with actin binding proteins（ABPs）to form highly ordered polymerization structure on the basis of F-actin,which plays a variety of important physiological functions.According to the full length cDNA of Dendrorhynchus zhejiangensis actin,the prokaryotic expression plasmid for actin（pET-28a-A）was constructed and transformed into E.coliBL21（DE3）.After IPTG induction,the recombinant proteins were highly expressed and contained in inclusion bodies.The recombinant proteins from inclusion bodies were purified by Nickel nitrilotriacetic（Ni-NTA）agarose affinity chromatography,and further dialyzed into a refolding buffer.The results of sodium dodecyl sulfate polycrylamide gel（SDS-PAGE）showed that the relative molecular weight of r-actin was 43 kD.In in vitro polymerization condition,the atomic force microscope（AFM）was used to observe and analyze the large-scale aggregate structure of r-actin during self-assembly.Our results showed that in vitro assembly of r-actin polymerized into complicated discrete structures,like filopodia-like fiber bundles,random coiled actin clusters,in addition to form disorganized protein aggregates.These data implicate that in vitro assembly of r-actin reflects its inherent thermodynamic properties of polymerization;further study would help to explore the regulatory function of actin binding proteins（ABPs）in the assembly of actin supramolecular aggregate structures.

关 键 词：浙江枝吻纽虫 肌动蛋白 原核表达 AFM 自组装 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]