Site-Specific And Efficient Incorporation Of UNAAs Based On Robust Cell-Free Protein Synthesis System

20 natural amino acids provide diverse variabilities on structures and functions for natural proteins.However,the increasing need for researches on protein engineering and protein-based drug fabrication could not be met based on natural proteins.The incorporation of unnatural amino acids(UNAA)with unique functional groups into proteins provides novel physicochemical properties for more diverse functionalities,including advanced pharmacokinetics,cancer therapeutics,vaccine development,proteomics and protein engineering.Traditional methods of UNAA incorporation in vivo requires living cells and transmembrane transport with limited type of incorporated UNAA and low incorporation efficiency.To solve these problems,it is necessary to develop a platform of UNAA corporation with adjustable components and no limitation of cell membrane.Therefore,cell-free protein synthesis(CFPS)platform has been considered as an effective platform for unnatural protein synthesis.Amber suppression is the most widely used for UNAA incorporation in CFPS.However,amber suppression could only achieve the incorporation of one type of UNAA.To achieve incorporation of different type of UNAAs,other stop codon suppression methods are needed simultaneously.In this paper,the reaction conditions of CFPS system,the establishment of cell-free unnatural protein synthesis system(CFUPS)based on multiple stop codons suppression,and the incorporation of different UNAAs were studied.The results of the study are as follows:(1)The effect of reporter protein,metal ion and crowding agent on the expression efficiency of CFPS protein was explored,the sf GFP was used as reporter protein,Mg²⁺was metal ion,and the high-efficiency CFPS system without crowding agent was used.The optimized system was used for therapeutic protein expression.(2)The target protein template and three OTS components were designed and prepared for stop codons suppression.Then,the redox environment,Mg²⁺concentration,and OTS optimum concentration were screened.The redox environment showed no significant effect on the synthesis of unnatural proteins.The optimal Mg²⁺concentrations were 30 mM,40 mM,and 35 mM;the optimal tRNA concentrations were 75 ng/?L,75 ng/?L,and 10 ng/?L;the optimal aa RS concentrations were 0.06mM,0.1 mM,0.3 mM;the optimal UNAA concentrations were 2 mM,2.5 mM,2mM for TAG,TAA,and TGA suppression,respectively.Unnatural sf GFP was expressed in the optimal system and characterized by mass spectrometry.TAG and TAA suppression of UNAA incorporation at specific sites were detected by mass spectrometry.The CFUPS system based on TAG and TAA suppression were successfully established.(3)The target protein template was prepared for multiple UNAAs incorporation,and the concentrations of Mg²⁺and OTS in CFUPS were screened.The optimal Mg²⁺concentration of the system was 30mM,and the optimal concentration of OTS compositions were:0.02 mM pPaFRS,25 ng/?L tRNA_CUA,2.5 mM pPaF,0.05 mM p Az FRS,25 ng/?L tRNA_UUA and 2.5 mM p Az F.The unnatural sf GFP synthesized by this system was characterized by mass spectrometry,and the site-specific incorporation of two UNAAs was successfully detected.The research aims to establish a more efficient CFUPS platform,further expand the functions and types of unnatural proteins,and promote the development and application of the CFUPS system.