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相关概念视频

Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
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人类ATE1 arginyltransferase的复合表达,净化和表征

Thilini Abeywansha1, Abigail Kim2, Sahil Bhaskaran1

  • 1Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH, United States.

Methods in enzymology
|August 31, 2025
PubMed
概括

本研究详细介绍了一种简单的生产和净化人类Arginyl-tRNA-蛋白转移酶1 (ATE1) 酶的方法. 这种优化的过程产生了高度纯净,活跃的ATE1,对于了解蛋白调节至关重要.

关键词:
其他类型化基转移酶翻译后的修改它们是:

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科学领域:

  • 生物化学
  • 分子生物学
  • 酵素学

背景情况:

  • 阿尔基尼-tRNA-蛋白转移酶1 (ATE1) 是参与蛋白质降解和调节的关键酶.
  • 了解ATE1的功能需要可靠的表达和净化方法.
  • 现有的方法可能无法有效地获得大量的活性酶.

研究的目的:

  • 开发一种简单有效的方法来从大肠杆菌中表达和净化人类复合ATE1.
  • 允许生产高纯度和酶活性ATE1的毫克级数量.
  • 建立可靠的测试以验证ATE1活性并量化其与tRNA的相互作用.

主要方法:

  • 人类ATE1在大肠杆菌中的重组表达.
  • 染色学净化技术以达到高纯度 (>98%).
  • 酶化试验与质谱学相结合用于活性验证.
  • 用于量化ATE1-tRNA结合 afinity 的电泳移动性转移试验 (EMSA).

主要成果:

  • 成功表达和净化了几毫克的人类ATE1.
  • 对于分离的ATE1酶,其纯度超过98%.
  • 通过化试验证明了纯化的ATE1的酶活性.
  • 使用EMSA量化ATE1-tRNA结合亲和力.

结论:

  • 这种方法提供了一种高效可靠的方法来获得高纯度,活跃的人类ATE1.
  • 该协议有助于进一步研究ATE1在蛋白调节和循环中的作用.
  • 经过验证的测试是特征ATE1功能和相互作用的重要工具.