Efficient Incorporation Of Unnatural Amino Acids Based On Escherichia Coli Cell-free Unnatural Protein Synthesis System

The cell-free unnatural protein synthesis(CFUPS)system serves as a powerful complement platform for expanding the structure and function of proteins,and has been successfully applied in basic scientific research and industrial production.The CFUPS system relies on crude cell extracts rather than living cells,breaking through the barrier of the cell membrane,enhancing the flexibility of the system for engineering design and fine-tuning of the reaction process.In addition,incorporating unnatural amino acids(UNAAs)with novel functionality can improve properties of the protein,such as post-translational modification of eukaryotic proteins,introduction of orthogonal reaction handles and biophysical probes,and polyprotein materials.At present,the methods of UNAA incorporation developed in CFUPS systems can be divided into two categories.One is global suppression based on natural translation system.The other contains stop codon suppression,frameshift suppression,reassignment of sense codons and unnatural base pairs based on orthogonal translation system.The most widely used one is the amber suppression method.This paper first systematically reviewed the advances of the methods for UNAAs incorporation in CFUPS systems and main applications of unnatural proteins.Then it analyzed the opportunities and challenges of the development of the system,such as low embedding efficiency and the difficulty in biomanufacturing.Secondly,established the CFUPS platform,by selecting proper reporter proteins,screening chassis cell,and optimizing the reaction conditions,the plasmid template concentration and orthogonal translation components,aiming to achieve high UNAA incorporation efficiency and high unnatural protein expression with minimal toxicity,as follows.1)The sfGFP was the optimum reporter protein,and the optimal condition of the reaction was 30? for 16 h.2)E.coli Rosetta(DE3)was the best universal cell.The efficiency of UNAA incorporation can be reach to 60%.3)The plasmid template should be added at 600~800 ng.The optimal concentrations of the four OTSs were: 75 ng/?L o-t DNA,0.04 mg/m L p Pa FRS and 5 m M p Pa F,0.5mg/m L.p Az FRS and 2.5 m M p Az F,0.5 mg/m L p Ac FRS and 2.5 m M p Ac F,0.3mg/m L p Bp FRS and 10 m M p Bp F.Then,the factors were studied which affected the efficiency of UNAA incorporation,including the effect of genomic engineering of the chassis cells,such as TAG mutation and knockout of the prf A encoding RF1,effect of different incorporation sites on the peptide chain,the fourth base effect and the rare codon effect,and orthogonality of OTS.Details as follows.1)By recoding the genome of chassis cells to dispel the competition between o-t RNA and RF1(such as E.coli C321.?A),100% UNAA incorporation efficiency and 1.0 mg/m L unnatural sf GFP with single UNAA can be achieved.2)The distance between the UNAA incorporation site in the peptide chain and the N/C terminal had no correlation with the efficiency of UNAA incorporation.3)When the fourth base was a purine nucleotide(A,G),it is more beneficial for UNAA incorporation than a pyrimidine nucleotide(T,C).4)When there were 1 or 2 rare codons before the incorporation site,the incorporation would be inhibited.Moreover,the effect was more significant when two rare codons were present.Finally,the incorporation of multiple UNAAs in CFUPS system was explored and had made some efforts to improve the expression of unnatural proteins by the addition of RNase or protease inhibitors.And 0.2 mg/m L of sf GFP containing 4 UNAAs was expressed.