Application Of Synthetic Coding Un-Natural Amino Acid Technology In The Long-lasting Modification Of Protein And Peptide Drugs

Compared with traditional chemical drugs,protein and peptide drugs have higher safety and target specificity,but there are still many challenges in their development and application.One of the most important shortcomings is that the half-life of proteins and peptide drugs is generally shorter in vivo.In order to overcome the above problems,researchers have gradually developed a variety of long-lasting protein technology to affect the rate and mechanism of drug metabolism in vivo by changing the structure of drug molecules,thereby extending the half-life of protein and peptide drugs.Compared with glycosylation modification,fusion protein modification and PEG modification,fatty acid modification is getting more attention from many researchers because it not only can achieve a long half-life,but also help to improve the fat-solubility,intestinal mucosal permeability and absorption efficiency of drugs.The traditional fatty acid modification of protein drugs is mainly achieved through chemical coupling in vitro,which has many limitations such as limited reaction conditions,low modification efficiency,complicated separation steps,and poor uniformity of modification.Synthetic coding technology utilizes orthogonal aminoacyl-tRNA synthetase to introduce modified amino acids(un-natural amino acids)in the host body to a specific site of the protein to produce a modified protein,so as to avoid the limitation to modify the protein in vitro.In this project,we designed an un-natural amino acid(HepoK)with 7 carbon atoms in fatty acid chain.An orthogonal aminoacyl-tRNA synthetase specific for HepoK was obtained via high-throughput screening.HepoK was introduced at specific sites of myoglobin,glucagon-like peptide 1(GLP-1)and fibroblast growth factor 21(FGF21)in E.coli and these fatty acid-modified proteins were separated and purified.Mass spectrometric analysis of myoglobin with HepoK confirms the specificity of HepoK insertion.Furthermore,comparison of interactions of fibroblast growth factor 21 and glucagon-like peptide 1 before and after HepoK insertion with human plasma albumin revealed that the protein with HepoK has stronger interaction with human plasma albumin.Moreover,binding of glucagon-like peptide 1 having Hepok to human plasma albumin is stronger than that of commercially available Liraglutide peptide.It is also proved that the fatty acid modified protein produced by synthetic encoding of unnatural amino acid can be combined with HSA in order to prolong its half-life in vivo.In conclusion,this study used synthetic coding technology for the first time to directly achieve protein fatty acid modification in the expression host and demonstrated that the modified protein specifically binds to human plasma albumin,thereby providing more efficient and uniform method for the production of long-lasting protein.