豌豆肌动蛋白异型体PEAc3的原核表达及其聚合特性分析

Prokaryotic expression and polymerization properties analysis of a pea actin isoform3(PEAc3)

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pET30 a-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pET30 a-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0.5μmol/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。

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.