| 摘要: |
| 植物肌动蛋白在植物顶端生长、细胞分裂分化和细胞信号转导等多种生命活动中发挥着重要作用。豌豆中存在多种肌动蛋白异型体。研究豌豆肌动蛋白异型体PEAc3与组氨酸标签(His-tag)及绿色荧光蛋白(GFP)的融合表达,并分析了融合蛋白的聚合特性。采用RT-PCR的方法克隆PEAc3基因,构建原核表达载体pET30a-His-PEAc3-GFP。用DNAman生物学软件分析表明,PEAc3融合蛋白全长675个氨基酸,分子量74.74 ku,等电点5.81。将pET30a-His-PEAc3-GFP转入大肠杆菌BL21中,优化的诱导表达条件为:25 ℃,当菌液OD600达到0.8时加入IPTG(浓度0.1 mmol/L),诱导表达4 h。采用尿素变性复性、镍柱亲和层析的方法从包涵体中纯化获得高纯度融合蛋白。融合蛋白His-PEAc3-GFP能够体外聚合,聚合临界浓度为0.5 μmol/L。单体His-PEAc3-GFP对DNaseⅠ有抑制作用,聚合后对肌球蛋白Mg-ATPase活性有一定的激活作用。上述结果表明,原核表达的His-PEAc3-GFP可能具有类似于一般肌动蛋白的聚合特性。 |
| 关键词: 豌豆 肌动蛋白 异型体 诱导表达 聚合特性分析 |
| DOI：10.11841/j.issn.1007-4333.2010.03.001 |
| 投稿时间：2009-11-30 |
| 基金项目:植物生理生化国家重点实验室开放课题(PPB003); 辽宁省教育厅项目(20080663); 沈阳农业大学中青年导师基金项目(2007001)第一作者:张少斌,副教授,主要从事蛋白质结构与功能研究,E-mail:zsb20022001@yahoo.com.cn通讯作者:刘国琴,教授,博士生导师,主要从事植物细胞骨架研究,E-mail:liu@cau.edu.cn |
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| Prokaryotic expression and polymerization properties analysis ofa pea actin isoform3 (PEAc3) |
|
ZHANG Shao-bin1, LIU Xi1, LIU Guo-qin2
|
| (1.Biological Science and Technology College,Shenyang Agricultural University,Shenyang 110161,China;2.National Key Laboratory of Plant Physiology and Biochemistry,China Agricultural University,Beijing 100193,China) |
| Abstract: |
| Plant actins play very important role in many living process,such as apical growth,cell division or differentiation,signal transduction.There are many actin isoforms in pea.In this paper,PEAc3 fused with His-tag and green fluorescence protein (GFP) was expressed in E.Coli,and the polymerization properties of the fusion protein were analyzed.The gene of PEAc3 was cloned by RT-PCR,and a prokaryotic expression vector of pET30a-His-PEAc3-GFP was constructed.The fusion protein has 674 amino acids,the molecular weight (MW) of fusion protein is 74.74 ku,and its isoelectric point is 5.81(predicted by DNAman).The plasmid of pET30a-His-PEAc3-GFP was transformed into E.coli strain of BL21,the induction expression conditions for the fusion protein is as following:the E.coli BL21 with plasmid was cultured at 25 ℃,followed by addition of IPTG(0.1 mmol/L) in the culture liquid when its OD600 was about 0.8.The induction was performed for four hours.The fusion protein of His-PEAc3-GFP was purified from inclusion body by Ni-resins chromatography after denaturation and renaturation in urea.The polymerization property analysis in vitro indicated that His-PEAc3-GFP could be polymerized.The critical concentration for polymerizing was 0.5 μmol/L.The His-PEAc3-GFP monomers could inhiit the activity of DNaseⅠ.The polymerized His-PEAc3-GFP had the ability of activating the Mg-ATPase of myosin.So,we could probably draw a conclusion that the His-PEAc3-GFP purified from E.coli. has the similar polymerization characteristics with that of normal actins. |
| Key words: pea actin isoform induction expression polymerization analysis |
