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

作　　者：Jiaqi Hou Xinjie Chen Nan Jiang Yanan Wang Yi Cui Lianju Ma Ying Lin Yuan Lu 

机构地区：[1]College of Life Sciences,Shenyang Normal University,Shenyang,110034,China [2]Key Laboratory of Industrial Biocatalysis,Ministry of Education,Department of Chemical Engineering,Tsinghua University,Beijing,100084,China [3]Guangdong Key Laboratory of Fermentation and Enzyme Engineering,School of Biology and Biological Engineering,South China University of Technology,Guangzhou,510006,China

出　　处：《Synthetic and Systems Biotechnology》2022年第1期522-532,共11页合成和系统生物技术（英文）

基　　金：the National Key R&D Program of China(Grant No.2018YFA0901700);the National Natural Science Foundation of China(Grant No.21878173);the Beijing Natural Science Foundation(Grant No.2192023).

摘　　要：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.

关 键 词：Cell-free protein synthesis Orthogonal translation system Unnatural protein Unnatural amino acid Terminal codon suppression 

分 类 号：O62[理学—有机化学]