Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNA_(CUA) Pair and Click Chemistry

摘要

The site-specific and homogeneous modification of recombinant proteins under physiological conditions is an important challenge. Cysteines and other amino acid residues in proteins can be specifically labeled by several methods, but site-specificity, as opposed to residue specificity, is difficult to achieve.rnSeveral phenylalanine derivatives can be site-specifically introduced into recombinant proteins in response to an amber codon inserted into the corresponding gene using an evolved tyrosyl-tRNA synthetase/tRNA~CUA pair that is orthogonal in E. coli. Phenylalanine derivatives bearing alkynyl, azido, and keto groups, that are bio-orthogonal in their chemical reactivity have been incorporated. However the introduction of aromatic amino acids at the majority of sites in proteins where aliphatic amino acids are naturally found may cause misfolding or loss of protein function; therefore there is a pressing need for methods to site-specifically incorporate aliphatic amino acids that contain bio-orthogonal chemical handles for use in protein labeling.