从部分随机的非催化脚手架中选择和进化酶
Burckhard Seelig1, Jack W Szostak
1Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology (CCIB), 7215 Simches Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA.
Nature
|August 19, 2007
概括
研究人员使用信使RNA显示器和天真蛋白质库创建了新的酶. 这种方法绕过了机械学知识的需求,使得高效率的新酶功能的发现成为可能.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 酵素工程是什么意思 酵素工程
背景情况:
- 酶是高效的生物催化剂,对于化学合成和制药至关重要.
- 开发新的酶往往需要对反应机制的广泛知识.
- 现有的酶改进方法包括选,选择和定向进化.
研究的目的:
- 为了证明真正新的酶活性在没有先前的机理信息的情况下 de novo 创建.
- 利用信使RNA显示器从一个庞大的,天真的库中选择功能性蛋白质.
- 进化具有优化的催化性能的新型RNA连接酶.
主要方法:
- 使用信使RNA显示器从原始蛋白质库 (>10^12序列) 中选择功能性蛋白质.
- 蛋白质与其编码mRNA的体外翻译和共价链接.
- 实验室定向进化适用于孤立的新型RNA连接酶.
- 使用产品形成作为唯一的选择标准.
主要成果:
- 从基于指支架的图书馆成功分离了新的RNA连接酶.
- 在进化的酶中实现了显著的速率提升 (>200万倍).
- 在工程酶中展示了多重的周转能力.
- 在不依赖机械学数据的情况下验证了de novo酶设计方法.
结论:
- 通过从多样化,天真的蛋白质库中进行选择,可以 de novo 创建真正新的酶活性.
- 使者RNA显示是体外蛋白质进化的强大技术,克服了体内限制.
- 这种方法加速了各种应用的酶的发现和优化.
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