In vitro selection for catalytic activity with ribosome display
In vitro selection for catalytic activity with ribosome display
复制标题
DOI：
10.1021/ja025870q
复制
发表时间：
2002-08-14
影响因子：
15
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Amstutz, P;Pelletier, JN;Plückthun, A
据我们所知，我们报道了第一次基于催化周转的体外催化活性选择，使用核糖体展示，这是一种在任何步骤都不涉及活细胞的方法。RTEM-β-内酰胺酶在核糖体上功能上与其编码的mRNA形成复合体。我们设计并合成了一种基于机理的β-内酰胺酶抑制剂--生物素化氨苄西林砜，适用于核糖体展示的β-内酰胺酶的催化活性的选择。氨苄西林的这种衍生物以一种特定的和不可逆转的方式灭活了β-内酰胺酶。在适当的选择条件下，活性RTEM-β-内酰胺酶相对于失活的点突变株在每个核糖体展示选择循环中得到100倍以上的丰富。用β-内酰胺酶抑制蛋白(BLIP)进行结合选择，得到了与自杀抑制剂选择相似的结果，表明核糖体展示在催化活性和亲和力选择方面具有类似的效率。在未来，使用完全体外过程直接选择酶活性的能力可能允许可探索序列空间相对于现有策略的显著增加。
We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated P-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.