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 , often rely on complex reaction buffers containing up to 35 components, limiting their widespread adoption and systematic optimization. Here, we present an optimized E. coli cell-free protein synthesis ( e CFPS) 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 e CFPS. This optimized and user-friendly e CFPS efficiently synthesizes challenging proteins, including functional, self-assembling vimentin, and active restriction endonuclease Bsa I despite its strong cytotoxicity, and serves as a powerful tool that will facilitate diverse applications in basic life science research and beyond.