Site-Specific Incorporation of p-Propargyloxyphenylalanine in a Cell-Free Environment for Direct Protein−Protein Click Conjugation

摘要: The tyrosine analog p-propargyloxyphenylalanine (pPa), like tyrosine, has limited water solubility. It been postulated that this solubility contributed to reduced cellular uptake of pPa and thus in vivo incorporation into proteins. Using a cell-free protein synthesis system (CFPS) circumvent uptake, incorporated site-specifically proteins with high specificity at yields up 27 times greater than the highest previously reported yield. alkyne group present on provides reactive residue for site-specific bioconjugation copper(I)-catalyzed azide−alkyne [3 + 2] cycloaddition (CuAAC). Previously, another CuAAC-compatible unnatural amino acid p-azido-l-phenylalanine (pAz) was demonstrated CFPS. However, may be preferred over pAz due instability pAz’s aryl-azido moiety upon UV or near-UV light exposure. Also, ability incorporate site-specifical...