Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis,ACS Synthetic Biology

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Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-07-16 , DOI: 10.1021/acssynbio.1c00195
Logan R Burrington _{1,

2} , Katharine R Watts _{1,

2} , Javin P Oza _{1,

2}

Affiliation

Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in high-yielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across in-house E. coli extracts and commercial kits. We then set out to fine-tune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30–50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.

中文翻译：

表征和改进基于大肠杆菌的无细胞蛋白质合成的反应时间

无细胞蛋白质合成 (CFPS) 是一种平台生物技术，能够按需合成各种应用的蛋白质。许多进步提高了 CFPS 平台的生产力，从而产生高产反应；然而，由于反应时间长，许多应用仍然受到限制。为了克服这一限制，我们首先建立了跨内部大肠杆菌的 CFPS 基准反应时间提取物和商业试剂盒。然后，我们着手对我们的内部提取系统进行微调，以缩短反应时间。通过优化反应组成和滴定低成本添加剂，我们确定了将反应时间缩短 30-50% 以获得用于生物制造应用的高蛋白滴度的配方，并将时间缩短 50% 以上以达到 sfGFP 检测限用于教育和诊断。在最佳条件下，我们在不到 10 分钟的时间内报告了 sfGFP 信号的可见观察。总之，这些进步增强了 CFPS 作为快速、用户定义平台的实用性。

更新日期：2021-08-20

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