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

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 113-118.

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

李晔¹, 陈蕾¹, 周君¹, 李成华¹, 苏秀榕¹, 李太武²

（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）

收稿日期:2013-04-02 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:李晔，博士，讲师，研究方向：生化与分子生物学，食品科学与工程；E-mail：liye@nbu.edu.cn
基金资助:
浙江省自然基金资助项目（Y3100480），浙江省教育厅科研项目（20070951）

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

Li Ye¹, Chen Lei¹, Zhou Jun¹, Li Chenghua¹, Su Xiurong¹, Li Taiwu²

（1. School of Marine Sciences，Ningbo University，Ningbo 315211；2. Ningbo City College of Vocational Technology，Ningbo 315100）

Received:2013-04-02 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

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.coli BL21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。

关键词: 浙江枝吻纽虫, 肌动蛋白, 原核表达, AFM, 自组装

Abstract: 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.coli BL21(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.

Key words: Dendrorhynchus zhejiangensis, Actin Prokaryotic expression, Atomic force microscope, Self-assembly

李晔, 陈蕾, 周君, 李成华, 苏秀榕, 李太武. 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[J]. 生物技术通报, 2013, 0(8): 113-118.

Li Ye, Chen Lei, Zhou Jun, Li Chenghua, Su Xiurong, Li Taiwu. Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro[J]. Biotechnology Bulletin, 2013, 0(8): 113-118.

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