豌豆肌动蛋白异型体（PEAc3）与绿色荧光蛋白（GFP）的融合表达与生化特性分析
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摘要
肌动蛋白普遍存在于真核生物中,在各种肌动蛋白结合蛋白的调控下,参与多种细胞活动,包括细胞分裂、内吞作用、成核作用、细胞信号传导、重力感应及顶端生长、细胞器运动等。在植物细胞中,由多基因家族编码的肌动蛋白存在多种异型体,不同肌动蛋白异型体的表达具有组织器官特异性,在植物生长发育过程中发挥着不同的功能。为了深入了解特定肌动蛋白异型体的功能及其动态调节,本研究利用GFP融合技术对豌豆肌动蛋白异型体3(PEAc3)进行了原核表达与纯化,并研究了其部分生物化学特性,主要研究结果如下：
利用RT-PCR的方法克隆了豌豆肌动蛋白异型体PEAc3基因,与随机挑选的7种其他植物肌动蛋白的同源性在90%以上。利用DNAman(Ver6.0)生物信息学软件,建立了豌豆种内异型体之间的生物进化树,为研究真核生物分子进化关系提供依据。根据预测的PEAc3氨基酸序列,推定的PEAc3蛋白MW为41.67kD,pI为5.17,可溶性蛋白没有信号肽序列,以成熟蛋白形式存在,含有肌动蛋白典型的三组序列指纹图谱,推测的PEAc3三维结构图与已有的动物骨骼肌肌动蛋白三维结构相似。
为了研究基因融合对PEAc3原核表达的影响,分别构建了四种原核表达载体PEAc3-pET30a, His-PEAc3-pET30a, His-PEAc3-GFP-pET30a和His-GFP-pET30a。利用DNAman软件分析上述载体表达的蛋白质,PEAc3全长为377个氨基酸,MW为41.67kD,pI为5.17；His-PEAc3全长为427个氨基酸,MW为47.13kD,pI为5.56；His-GFP-PEAc3全长为675个氨基酸,MW为74.74kD,pI为5.81；His-GFP全长为296个氨基酸,MW为33.04kD,pI为6.26。利用电穿孔法将构建的载体转入大肠杆菌BL21中,对蛋白的诱导表达条件进行了优化：PEAc3添加IPTG诱导表达的最适菌液浓度为OD600=0.7,IPTG浓度为0.05mmol/L,诱导表达时间为4h,温度为25℃,主要在包涵体中表达；His-PEAc3在菌液浓度为OD600=0.5时添加IPTG,浓度为O.lmmol/L,诱导表达时间为3h,温度为37℃,主要在包涵体中表达；His-PEAc3-GFP在菌液浓度为OD600=0.8时添加IPTG,浓度为0.1mmol/L,诱导表达时间为4h,温度为25℃,在上清和包涵体中都有表达；His-GFP在菌液浓度为OD600=0.5时添加IPTG,浓度为0.05mmol/L,诱导表达时间为4h,温度为37℃,主要在上清中表达。上述结果表明,GFP融合对于PEAc3在原核体系中的可溶性表达有一定的促进作用。
利用尿素变性复性,镍柱亲和层析、离子交换层析等方法,分别纯化了上述四种蛋白,并研究了其部分生化特性。通过比较PEAc3、His-PEAc3和His-PEAc3-GFP的聚合曲线和聚合临界浓度,发现His-PEAc3-GFP的聚合动力学与动物骨骼肌肌动蛋白更为接近。DNase I活性测定表明,PEAc3, His-PEAc3和His-PEAc3-GFP对DNase I都有抑制作用,其中His-PEAc3-GFP的抑制效果最明显。His-PEAc3-GFP对肌球蛋白Mg-ATPase活性具有明显激活效果,达到空白对照的五倍。上述研究表明,His和GFP的融合对PEAc3在原核系统中的正确表达与折叠有促进作用,GFP融合没有影响PEAc3的体外生化特性。
本课题对豌豆肌动蛋白异型体PEAc3基因进行克隆和表达,对其GFP融合蛋白的部分生化特性进行分析,研究结果对于深入了解高等植物肌动蛋白异型体,探索肌动蛋白结构和功能具有重要意义。利用GFP融合表达不仅可以获得大量有活性的植物肌动蛋白异型体,从而为体外的相关研究提供实验材料,同时利用GFP的绿色荧光,一方面可以方便体外的相关研究,另一方面为研究体内肌动蛋白异型体聚合解聚的动态变化奠定一定的基础。
Actins exist in eukaryotes generally. Under the control of various kinds of actin-binding proteins, actins participate in various cellular activities, such as cell division, endocytosis, nucleation, cell signaling, gravity sensing, and the tip growth, organelle movement etc. There are multiple actin isoforms which are encoded by the multi-gene family in plant cells; the actin isoforms expressed in tissue and organ specificity; and the actin isoforms play different functions in plant growth and development. In order to understand the function and dynamic regulation of the specific actin isoforms deeply, this study expressed and purified pea actin isoforms 3 (PEAc3) in prokaryotic expression system by GFP fusion technique and researched some biochemical characteristics of the fusionprotein, the main results are show as follows:
Pea actin isoforms PEAc3 gene was cloned by RT-PCR method and compared with the seven kinds of other plant actins selected randomly, the homology was above 90%. Using DNAman (Ver6.0) bioinformatics software, a biological evolutionary tree was established among the pea actin isoforms which provided the support for the study of molecular evolution within eukaryotic. Based on the predicted amino acid sequence of PEAc3, the molecular weight (MW) of PEAc3 was 41.67kD; the pI was 5.17; PEAc3 was soluble protein without signal peptide sequence and exist as a mature protein, PEAc3 have three groups of sequence fingerprint which is the typical feature of actin; The predicted 3D map of PEAc3 is similar with that of skeletal muscle actin.
In order to study the effect of gene fusion on PEAc3 prokaryotic expression, four prokaryotic expression vectors were constructed, that is PEAc3-pET30a, His-PEAc3-pET30a, His-PEAc3-GFP-pET30a and His-GFP-pET30a. The proteins expressed by above four vectors were analyzed by DNAman software:The length of PEAc3 was 377 amino acids, MW was 41.67kD, pI was 5.17; the length of His-PEAc3 was 427 amino acids, MW was 47.13kD, pI was 5.56; the length of His-GFP-PEAc3 was 675 amino acids, MW was 74.74kD, pI was 5.81; the length of His-GFP was 296 amino acids, MW was 33.04kD, pI was 6.26. The constructed vector was transformed into E. coli. BL21 by electroporation, the conditions of induced expression was optimized:PEAc3:the best bacterial concentration of adding IPTG was OD6oo=0.7, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 25℃, expressed in inclusion bodies mainly; His-PEAc3:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.1mmol/L, induced time was 3h, temperature was 37℃, expressed in inclusion bodies mainly; His-PEAc3-GFP:the best bacterial concentration of adding IPTG was OD6oo=0.8, and the optimal IPTG concentration was 0.1 mmol/L, induced time was 4h, temperature was 25℃, expressed in supernatant and inclusion bodies; His-GFP:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 37℃, expressed in supernatant mainly. The results showed that the fusion of GFP accelerated the soluble expression of PEAc3 in prokaryotic expression system.
The four proteins above were purified by the denaturation and renaturation of urea, nickel column affinity chromatography and ion exchange chromatography etc. and some biochemical characteristics of the proteins were studied. By the comparison of polymerization curve and the critical polymerization concentration of PEAc3, His-PEAc3 and His-PEAc3-GFP, the polymerization kinetics of His-PEAc3-GFP is closer to that of the skeletal muscle actin. Determination of DNaseⅠactivity showed that, PEAc3, His-PEAc3 and His-PEAc3-GFP could inhibit the activity of DNaseⅠ, the effect of His-PEAc3-GFP was the most obviously. The activation effect of His-PEAc3-GFP on myosin Mg-ATPase activity was very obviously, reaching above five times than that of the blank control. The study shows that, the fusion of His and GFP could promote the correct expression and folding of PEAc3 in prokaryotic system, the fusion of GFP did not affect the biochemical characteristics of PEAc3 in vitro.
The PEAc3 gene was cloned and expressed in E. coli. in this paper, and some biochemical properties of PEAc3 fusion with GFP were analysised, which was important for understanding higher plants actin isoforms deeply and studying the structure and function of actin. By GFP fusion expression system in this paper, we could not only gain abundant active plant actin isoforms for the relative research in vitro, but also study actins easily in vitro using the green fluorescent of GFP, and lay the foundation for the study of the dynamic changes of polymerization and depolymerization of actin iso forms in vivo.
利用RT-PCR的方法克隆了豌豆肌动蛋白异型体PEAc3基因,与随机挑选的7种其他植物肌动蛋白的同源性在90%以上。利用DNAman(Ver6.0)生物信息学软件,建立了豌豆种内异型体之间的生物进化树,为研究真核生物分子进化关系提供依据。根据预测的PEAc3氨基酸序列,推定的PEAc3蛋白MW为41.67kD,pI为5.17,可溶性蛋白没有信号肽序列,以成熟蛋白形式存在,含有肌动蛋白典型的三组序列指纹图谱,推测的PEAc3三维结构图与已有的动物骨骼肌肌动蛋白三维结构相似。
为了研究基因融合对PEAc3原核表达的影响,分别构建了四种原核表达载体PEAc3-pET30a, His-PEAc3-pET30a, His-PEAc3-GFP-pET30a和His-GFP-pET30a。利用DNAman软件分析上述载体表达的蛋白质,PEAc3全长为377个氨基酸,MW为41.67kD,pI为5.17；His-PEAc3全长为427个氨基酸,MW为47.13kD,pI为5.56；His-GFP-PEAc3全长为675个氨基酸,MW为74.74kD,pI为5.81；His-GFP全长为296个氨基酸,MW为33.04kD,pI为6.26。利用电穿孔法将构建的载体转入大肠杆菌BL21中,对蛋白的诱导表达条件进行了优化：PEAc3添加IPTG诱导表达的最适菌液浓度为OD600=0.7,IPTG浓度为0.05mmol/L,诱导表达时间为4h,温度为25℃,主要在包涵体中表达；His-PEAc3在菌液浓度为OD600=0.5时添加IPTG,浓度为O.lmmol/L,诱导表达时间为3h,温度为37℃,主要在包涵体中表达；His-PEAc3-GFP在菌液浓度为OD600=0.8时添加IPTG,浓度为0.1mmol/L,诱导表达时间为4h,温度为25℃,在上清和包涵体中都有表达；His-GFP在菌液浓度为OD600=0.5时添加IPTG,浓度为0.05mmol/L,诱导表达时间为4h,温度为37℃,主要在上清中表达。上述结果表明,GFP融合对于PEAc3在原核体系中的可溶性表达有一定的促进作用。
利用尿素变性复性,镍柱亲和层析、离子交换层析等方法,分别纯化了上述四种蛋白,并研究了其部分生化特性。通过比较PEAc3、His-PEAc3和His-PEAc3-GFP的聚合曲线和聚合临界浓度,发现His-PEAc3-GFP的聚合动力学与动物骨骼肌肌动蛋白更为接近。DNase I活性测定表明,PEAc3, His-PEAc3和His-PEAc3-GFP对DNase I都有抑制作用,其中His-PEAc3-GFP的抑制效果最明显。His-PEAc3-GFP对肌球蛋白Mg-ATPase活性具有明显激活效果,达到空白对照的五倍。上述研究表明,His和GFP的融合对PEAc3在原核系统中的正确表达与折叠有促进作用,GFP融合没有影响PEAc3的体外生化特性。
本课题对豌豆肌动蛋白异型体PEAc3基因进行克隆和表达,对其GFP融合蛋白的部分生化特性进行分析,研究结果对于深入了解高等植物肌动蛋白异型体,探索肌动蛋白结构和功能具有重要意义。利用GFP融合表达不仅可以获得大量有活性的植物肌动蛋白异型体,从而为体外的相关研究提供实验材料,同时利用GFP的绿色荧光,一方面可以方便体外的相关研究,另一方面为研究体内肌动蛋白异型体聚合解聚的动态变化奠定一定的基础。
Actins exist in eukaryotes generally. Under the control of various kinds of actin-binding proteins, actins participate in various cellular activities, such as cell division, endocytosis, nucleation, cell signaling, gravity sensing, and the tip growth, organelle movement etc. There are multiple actin isoforms which are encoded by the multi-gene family in plant cells; the actin isoforms expressed in tissue and organ specificity; and the actin isoforms play different functions in plant growth and development. In order to understand the function and dynamic regulation of the specific actin isoforms deeply, this study expressed and purified pea actin isoforms 3 (PEAc3) in prokaryotic expression system by GFP fusion technique and researched some biochemical characteristics of the fusionprotein, the main results are show as follows:
Pea actin isoforms PEAc3 gene was cloned by RT-PCR method and compared with the seven kinds of other plant actins selected randomly, the homology was above 90%. Using DNAman (Ver6.0) bioinformatics software, a biological evolutionary tree was established among the pea actin isoforms which provided the support for the study of molecular evolution within eukaryotic. Based on the predicted amino acid sequence of PEAc3, the molecular weight (MW) of PEAc3 was 41.67kD; the pI was 5.17; PEAc3 was soluble protein without signal peptide sequence and exist as a mature protein, PEAc3 have three groups of sequence fingerprint which is the typical feature of actin; The predicted 3D map of PEAc3 is similar with that of skeletal muscle actin.
In order to study the effect of gene fusion on PEAc3 prokaryotic expression, four prokaryotic expression vectors were constructed, that is PEAc3-pET30a, His-PEAc3-pET30a, His-PEAc3-GFP-pET30a and His-GFP-pET30a. The proteins expressed by above four vectors were analyzed by DNAman software:The length of PEAc3 was 377 amino acids, MW was 41.67kD, pI was 5.17; the length of His-PEAc3 was 427 amino acids, MW was 47.13kD, pI was 5.56; the length of His-GFP-PEAc3 was 675 amino acids, MW was 74.74kD, pI was 5.81; the length of His-GFP was 296 amino acids, MW was 33.04kD, pI was 6.26. The constructed vector was transformed into E. coli. BL21 by electroporation, the conditions of induced expression was optimized:PEAc3:the best bacterial concentration of adding IPTG was OD6oo=0.7, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 25℃, expressed in inclusion bodies mainly; His-PEAc3:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.1mmol/L, induced time was 3h, temperature was 37℃, expressed in inclusion bodies mainly; His-PEAc3-GFP:the best bacterial concentration of adding IPTG was OD6oo=0.8, and the optimal IPTG concentration was 0.1 mmol/L, induced time was 4h, temperature was 25℃, expressed in supernatant and inclusion bodies; His-GFP:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 37℃, expressed in supernatant mainly. The results showed that the fusion of GFP accelerated the soluble expression of PEAc3 in prokaryotic expression system.
The four proteins above were purified by the denaturation and renaturation of urea, nickel column affinity chromatography and ion exchange chromatography etc. and some biochemical characteristics of the proteins were studied. By the comparison of polymerization curve and the critical polymerization concentration of PEAc3, His-PEAc3 and His-PEAc3-GFP, the polymerization kinetics of His-PEAc3-GFP is closer to that of the skeletal muscle actin. Determination of DNaseⅠactivity showed that, PEAc3, His-PEAc3 and His-PEAc3-GFP could inhibit the activity of DNaseⅠ, the effect of His-PEAc3-GFP was the most obviously. The activation effect of His-PEAc3-GFP on myosin Mg-ATPase activity was very obviously, reaching above five times than that of the blank control. The study shows that, the fusion of His and GFP could promote the correct expression and folding of PEAc3 in prokaryotic system, the fusion of GFP did not affect the biochemical characteristics of PEAc3 in vitro.
The PEAc3 gene was cloned and expressed in E. coli. in this paper, and some biochemical properties of PEAc3 fusion with GFP were analysised, which was important for understanding higher plants actin isoforms deeply and studying the structure and function of actin. By GFP fusion expression system in this paper, we could not only gain abundant active plant actin isoforms for the relative research in vitro, but also study actins easily in vitro using the green fluorescent of GFP, and lay the foundation for the study of the dynamic changes of polymerization and depolymerization of actin iso forms in vivo.
引文
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