A Single Polyphosphate Kinase-Based NTP Regeneration System Driving Cell-Free Protein Synthesis

Current reconstituted cell-free protein synthesis systems regenerate the ATP and GTP required for peptide bond formation using phosphocreatine and three coupled kinase reactions. Here, we replace the three-kinase system with a single polyphosphate kinase, resulting in a comparable protein yield and enabling functional expression of a heat-sensitive enzyme at 37°C.