Translational Incorporation of Multiple Unnatural Amino Acids in a Cell-free Protein Synthesis System

摘要

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.