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肌动蛋白普遍存在于真核生物细胞中, 并在细胞生命活动中发挥重要作用. 在细胞中, 肌动蛋白多以异型体(isoform)形式存在. 植物肌动蛋白异型体的大量获得和理化特性分析一直是一个难题. 对豌豆肌动蛋白异型体PEAc1与组氨酸标签(His-tag)、绿色荧光蛋白(GFP)进行了基因融合和原核表达. 通过脲变性、梯度脲透析复性、镍柱亲和层析的方法从包涵体中纯化出大量、高纯度的PEAc1-GFP融合蛋白(> 2 mg/L培养物). 理化特性分析表明, 它同鸡骨骼肌肌动蛋白一样, 单体PEAc1-GFP能与DNaseⅠ 结合并显著抑制后者酶活性; 单体PEAc1-GFP能聚合成带有绿色荧光的丝状结构; 聚合的PEAc1- GFP能被微丝的特异标记物鬼笔环肽(phalloidin)标记; PEAc1-GFP与鸡骨骼肌肌动蛋白的聚合曲线趋势基本一致, 聚合临界浓度为0.24 mmol/L; 聚合的PEAc1-GFP能激活肌球蛋白Mg-ATPase活性, 其激活比率随浓度的增加而增加, 在1 mg/mL浓度下, 能激活4倍以上. 上述结果表明, 带有组氨酸标签和GFP标记的PEAc1表现出和天然肌动蛋白相似的理化特性; 基因融合、原核表达、变性、复性及亲和纯化是获得大量高纯度植物肌动蛋白异型体的有效方法.
Actin is ubiquitous in eukaryotic cells and plays an important role in the life-activity of cells, where actin is mostly in the form of isoforms.A large number of plant actin isoforms are obtained and physico-chemical Characterization has always been a challenge.The fusion and prokaryotic expression of pea actin isoforms PEAc1 and His-tag and green fluorescent protein (GFP) were studied.Urea denaturation, Purification of PEAc1-GFP fusion protein (> 2 mg / L in culture) from inclusion bodies by nickel column affinity chromatography showed that it was the same as chicken skeletal muscle actin Body PEAc1-GFP can bind to DNase Ⅰ and significantly inhibit the activity of the latter; Monomeric PEAc1-GFP can be polymerized into a filamentous structure with green fluorescence; Polymerized PEAc1-GFP can be detected by microcystin-specific marker phalloidin (phalloidin); PEAc1-GFP and chicken skeletal muscle actin polymerization trend of the same line, the critical concentration of polymerization 0.24 mmol / L; Polymerized PEAc1-GFP can activate myosin Mg-ATPase activity, the activation rate with Increase in concentration , Which can activate more than 4 times at the concentration of 1 mg / mL.The above results indicate that the histidine-tagged and GFP-tagged PEAc1 shows similar physico-chemical properties as native actin; gene fusion, prokaryotic expression, denaturation , Renaturation and affinity purification are effective methods for obtaining a large number of high-purity plant actin isoforms.