ABSTRACT
Cell-free unnatural protein synthesis (CFUPS) as an emerging approach for protein engineering research, has been successfully applied in basic scientific studies (e.g., protein-protein interaction, protein-nucleic acid interaction) and industrial production (e.g., pharmaceutical proteins, protein materials). CFUPS can improve the engineering freedom and the process control by allowing free addition of genetic elements and chemicals as research purposes. It also can give protein novel characteristics, structures and functions, including post-translational modification of proteins, incorporation of reaction handles, synthesis of biophysical probes and polymeric protein. This article systematically reviews the unnatural amino acid incorporation methods (global suppression, stop codon suppression, frameshift suppression and unnatural base-pairs) and the application advances of unnatural amino acids in protein modifications, biophysical probes, enzyme engineering, biomaterials and biopharmaceutical protein production. The opportunities and challenges of the CFUPS system development and the wide application of industrialization are also illustrated with details.