Cell-free protein synthesis (CFPS) technology serves as a powerful approach in synthetic biology, offering unique advantages such as an open, rapid, and programmable environment. It holds significant value across various applications, including mRNA translation research, high-throughput protein engineering, in vitro diagnostics, and biopharmaceutical preparation. However, high production costs and complex reaction components have historically posed substantial technical barriers. In this work, the team developed a highly streamlined Escherichia coli-based cell-free protein synthesis (eCFPS) system that significantly reduces system complexity while maintaining robust performance, establishing a highly accessible next-generation synthesis platform.

Conventional eCFPS systems typically require up more than 30 distinct reaction components, which often lead to potential inhibitory interactions among ingredients. Furthermore, the standard preparation of the core material—cell extract—is a laborious process that relies on extensive run-off reactions and dialysis, resulting in high costs, operational difficulties, and variable stability.

To address these challenges, we implemented a dual-simplification strategy to build an efficient eCFPS platform:

1. Through systematic and stepwise screening, the core essential components were streamlined down to just 7 key ingredients, significantly lowering production costs and eliminating potential inhibitory interactions.

2. The team developed a rapid extract preparation protocol that eliminates time-consuming steps. This method leverages endogenous metabolic enzymes and residual cellular components to compensate for the reduction in exogenous ingredients, substantially improving the overall workflow convenience.

Notably, this simplified design does not compromise system performance. The translational quality and yield of this streamlined system are comparable to or exceed those of conventional, highly complex systems. The platform successfully synthesized the inherently toxic endonuclease BsaI, as well as the aggregation-prone scaffold protein Vimentin, which successfully self-assembled into functional filaments. By lowering the entry barrier for CFPS technology, this work provides an efficient and accessible tool for synthetic biology and related industrial applications. The relevant methodologies have been filed for patent protection.

Paper Link: https://elifesciences.org/reviewed-preprints/109495