Centro de Biología Molecular "Severo Ochoa" (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.
Instituto de Biología Molecular, Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.
Microb Cell Fact. 2024 Jun 10;23(1):169. doi: 10.1186/s12934-024-02440-y.
In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.
Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.
We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.
Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
体外表达涉及在无细胞环境中利用细胞转录和翻译机制来产生一种或多种感兴趣的蛋白质，已在合成生物学和药物生物制造中得到广泛应用。大多数现有的体外表达系统在中等温度下活跃，但为了在高温下筛选具有高度热稳定性的酶或酶变体的天然或人工遗传多样性的大型文库，能够在高温下进行蛋白质合成是至关重要的。
开发一种与酶测定兼容的高温体外表达系统，并采用能够在减少体积中进行超高通量蛋白质表达的技术，例如微流控水包油（w/o）液滴。
我们从嗜热栖热菌中制备了用于体外翻译的无细胞提取物，其中包括用于能量再生的热稳定酶级联反应和用于转录的中温稳定 RNA 聚合酶，这最终限制了蛋白质合成的温度。产量与其他热稳定体外表达系统相当或更高，而制备程序要简单得多，并且可以适用于不同的嗜热栖热菌菌株。此外，这些提取物首次实现了在微流控液滴中进行高温体外表达。
嗜热栖热菌的无细胞提取物代表了一种比经过大量优化或纯组分热稳定体外表达系统更简单的替代方案。此外，由于它们与高温下的液滴微流控和酶测定兼容，所报道的系统代表了在更高温度下进行超高通量酶筛选的便捷途径。
