A simplified and highly efficient cell-free protein synthesis system for prokaryotes

Cell-free protein synthesis (CFPS) systems are a powerful platform with immense potential in fundamental research, biotechnology, and synthetic biology. Conventional prokaryotic CFPS systems, particularly those derived from Escherichia coli (E. coli), often rely on complex reaction buffers containing up to thirty-five components, limiting their widespread adoption and systematic optimization. Here, we present an optimized E. coli cell-free protein synthesis (eCFPS) system, which is significantly streamlined for high efficiency. Through systematic screening, we successfully reduced the essential core reaction components from 35 to a core set of 7. The thorough optimization of these seven key components ensured that protein expression levels were not only maintained but even substantially improved. Furthermore, we developed a much simpler procedure for preparing the bacterial cytosolic extracts, a “fast lysate” protocol that eliminates the traditional time-consuming runoff and dialysis steps, thereby enhancing the overall accessibility and robustness of eCFPS. This optimized and user-friendly eCFPS efficiently synthesizes challenging proteins, including functional, self-assembling vimentin, and active restriction endonuclease BsaI despite its strong cytotoxicity, and serves as a powerful tool that will facilitate diverse applications in basic life science research and beyond.