Mechanism investigation for efficient cell-free unnatural amino acid embedding

Cell-free unnatural protein synthesis (CFUPS) system, as a powerful unnatural protein synthesis platform, has been successfully developed from a small-batch laboratory scale to an industrialized production scale for various basic and applied research. However, to further promote the development and application of the CFUPS system, it is still necessary to optimize the unnatural amino acids (UNAAs) embedding system and explore the influencing factors. In this study, a typical CFUPS platform was first established and optimized. Based on this platform, the mechanism of UNAA incorporation was explored, including release factor 1 (RF1) effect, incorporation site effect, fourth base effect, rare codon effect, and orthogonal translation system (OTS) orthogonality. Furthermore, multisite UNAA incorporation was performed to synthesize unnatural proteins containing multiple UNAAs. In this optimized CFUPS system, the yield of green fluorescent protein (sfGFP) could reach 1306 mg/L, and the yield of modified sfGFP with single p-propargyloxy-L-phenylalanine (pPaF) could reach 804 mg/L. The modified sfGFP containing four pPaF could also be synthesized at a high yield of 197 mg/L. This study not only provides a theoretical basis for improving the incorporation efficiency of UNAAs, but also provides more possibilities for further expanding the types and functions of unnatural proteins.