条件蛋白质拼接:一种新的工具来控制蛋白质的结构和功能在体外和体外
Henning D Mootz1, Elyse S Blum, Amy B Tyszkiewicz
1Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Journal of the American Chemical Society
|August 28, 2003
概括
研究人员开发了一种有条件蛋白质拼接 (CPS) 系统,使用被拉帕米辛激活的分裂蛋白. 该系统使哺乳动物细胞中可控的蛋白质结合具有快速,剂量依赖的反应,为蛋白质工程提供了一个新的工具.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 合成生物学 合成生物学
背景情况:
- 蛋白质拼接是一种自然过程,在这种过程中,intins自剪除,加入侧面的exteins.
- 工程分裂蛋白质提供可控制的蛋白质结合.
- 拉帕米辛可以在工程分裂的VMA蛋白中触发拼接.
研究的目的:
- 为了证明条件蛋白拼接 (CPS) 系统在哺乳动物细胞中的实用性.
- 研究拉巴素诱导的蛋白质拼接的动力学和控制.
- 探索CPS在蛋白质组学中的灵活性和应用.
主要方法:
- 哺乳动物细胞的过渡性转染与编码构造物分裂的蛋白质半体融合到记者蛋白 (MBP和His-tag).
- 诱导蛋白质拼接使用拉巴或其类型.
- 通过西式涂抹和免疫沉检测拼接蛋白质产品.
- 使用ascomycin的剂量反应和竞争性抑制研究.
- 使用具有FKBP/FRB域的纯化蛋白质,研究几何灵活性.
主要成果:
- 在哺乳动物细胞中成功证明了条件蛋白拼接 (CPS).
- 拼接依赖于拉巴胺,快速 (10分钟内) 和剂量依赖.
- 在没有诱导剂的24小时内,没有观察到背景拼接.
- 阿斯科米辛可以竞争性地抑制拼接过程.
- CPS 组件显示了几何灵活性,FKBP/FRB 域融合到 extein 位置,仍然允许拉巴素触发的拼接.
结论:
- CPS系统是哺乳动物细胞中可控蛋白质结合的可行工具.
- 该系统提供对蛋白质结构和功能进行快速,可调节的控制.
- 在蛋白质组学和生物技术中,CPS具有广泛的潜在应用.
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