Insertion of Synthetic Peptides into Proteins by Tandem Protein Trans-Splicing

Abstract

Engineering proteins through chemical or genetic manipulations offers great potential to design and study proteins with complex properties. Despite tremendous progress, the type and combination of chemical modification (e.g. non-canonical amino acids (ncAAs) or post-translational modifications (PTMs)) that can be incorporated by genetic means (e.g. amber codon suppression) is still limited. Semi-synthetic approaches offer a post-translational alternative to synthetically introduce modifications into proteins, but have typically been performed in vitro and restricted to small proteins that can be easily refolded. Here, we present a method to incorporate synthetic peptides carrying multiple PTMs or ncAAs into both cytosolic and membrane proteins. The work is performed in live eukaryotic cells by tandem protein trans-splicing (PTS) using two orthogonal split intein pairs. We anticipate the approach to be broadly applicable as it allows for the introduction of a virtually limitless array of modifications, including those not accessible by existing methods.