Toward efficient multiple-site incorporation of unnatural amino acids using cell-free translation system

Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.