用非正规的氨基酸取代32个氨酸残留物会产生高度活跃的DNA聚合酶
Bastian Holzberger1, Andreas Marx
1Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraβe 10, 78457 Konstanz, Germany.
Journal of the American Chemical Society
|October 22, 2010
概括
蛋白质工程成功地将非正规氨基酸纳入了DNA聚合酶. 这种多化酶保留了关键功能,显示了未来蛋白质工程应用的前景.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 蛋白质工程是指蛋白质的工程.
背景情况:
- 蛋白质工程传统上使用20种天然氨基酸.
- 非正规氨基酸为蛋白质设计提供了新的功能.
- DNA聚合酶是动态酶,对于DNA合成至关重要.
研究的目的:
- 使用非正规氨基酸产生多化DNA聚合酶.
- 评估非正规氨基酸替代对酶活性,选择性和稳定性的影响.
- 探索非正规氨基酸在工程高度动态酶中的潜力.
主要方法:
- 在KlenTaq DNA聚合酶中,将32个林残留物与 (4R) - 罗林进行全球替换.
- 使用原料延伸和PCR测定来研究酶活性.
- 将化酶与父母野生类型酶进行比较.
主要成果:
- 实现了用 (4R) - 罗林有效替代 (92%) 的林残留物.
- 由此产生的化DNA聚合酶表现出高活性,保持忠实度和灵敏度.
- 与野生类型相比,该酶的热稳定性略有下降.
结论:
- 在大型,动态的酶中,非正规的氨基酸替代是可行的,而不会影响基本性质.
- 化DNA聚合酶作为一种有价值的工具和进一步蛋白质工程的起点.
- 这项工作通过展示非正规氨基酸的实用性来扩大蛋白质工程的工具包.
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