New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2010 Jul;38(13):e141. doi: 10.1093/nar/gkq377. Epub 2010 May 10.
In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes.
体外重建生物复合物或过程通常涉及多个单独纯化的蛋白质组件的组装。在这里，我们提出了一种策略，该策略将多个基因产物的表达和组装与体外重组蛋白合成系统中的功能检测相结合。该策略仅使用 DNA 模板而不是纯化的蛋白质，就有可能对多组分生物复合物或过程进行实验重建。我们将该策略应用于细菌转录起始，通过在体外重组蛋白合成系统中共同表达编码大肠杆菌 RNA 聚合酶亚基和 sigma 因子的基因，并通过将功能性 RNA 聚合酶全酶的合成和组装与报告基因的表达偶联，来实现这一点。使用这样的系统，我们证明了依赖于 sigma 因子的、启动子特异性的转录起始。由于蛋白质合成、复合物形成和酶催化都发生在同一个体外反应混合物中，因此这个重建过程类似于天然生物合成途径，并且避免了单个蛋白质的耗时表达和纯化。该策略可以显著减少传统重建方法通常所需的时间，允许快速生成和检测遗传突变，并为体外研究和干预复杂生物过程提供一个开放和可设计的平台。
