Abstract
Since the cell-free protein synthesis system is not limited by the cell growth, all the substrates are used to produce the protein of interest, and the reaction environment can be flexibly controlled. All the advantages allow it to synthesize toxic proteins, membrane proteins, and unnatural proteins that are difficult to make in vivo. However, one typical reason why the cell-free system has not been widely accepted as a practical alternative, is its expression efficiency problem. The Escherichia coli-based system was chosen in this study, and the model protein deGFP was expressed to explore a more efficient cell-free system. The results showed that Mg2+ with a concentration of 15 mM in the cell-free system with BL21 Star (DE3) as the extract could better synthesize protein. The smaller the vectors, the lighter the burden, the higher the protein synthesis. Simulating the crowding effect in the cell does not improve the protein expression efficiency of the optimized cell-free protein synthesis system. Based on the optimized system, the cell-free fundamental research platform, primary screening platform, and portable biomolecular synthesis platform were established. This study provides a robust cell-free protein synthesis toolbox with easy extract preparation and high protein yield. It also enables more researchers to reap the benefits from the cell-free biosynthesis platform.