...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Cell Research >Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1).
【24h】

Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1).

机译：GFP融合的豌豆肌动蛋白亚型（PEAc1）的可溶性表达和表征。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating SepharoseTM Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization, and activated the myosin Mg-ATPase activities after polymerization. The PEAc1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 uM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAc1-GFP was also expressed in tobacco BY2 cells, which offers a new pathway for further studying its distribution and function in vivo.
机译：具有绿色荧光蛋白（GFP）融合至其C末端和His-tag融合至其N末端的豌豆肌动蛋白同种型PEAc1以可溶形式在原核细胞中表达，并通过Ni-螯合SepharoseTM Fast Flow色谱柱进行了高度纯化。纯化的融合蛋白（PEAc1-GFP）在聚合前有效抑制DNase I活性，并在聚合后激活肌球蛋白Mg-ATPase活性。 PEAc1-GFP也聚合成临界浓度为0.75 uM的绿色荧光丝状结构。这些丝状结构被TRITC-鬼笔环肽标记，肌动蛋白微丝染色的特异性试剂，经负染色鉴定为直径为9 nm。这些结果表明，即使具有His标签和GFP融合，PEAc1仍保留了肌动蛋白的基本特征，表明使用GFP融合蛋白获得可溶性植物肌动蛋白同工型以在体外分析其物理和生化特性的潜在潜力。 PEAc1-GFP也在烟草BY2细胞中表达，为进一步研究其在体内的分布和功能提供了新途径。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号