(534d) A Cell-Free Approach to Engineering the Site-Specific Incorporation of Unnatural Amino Acids for Direct Protein-Protein Conjugation

We have engineered a cell-free protein synthesis system for site-specific incorporation of unnatural amino acids to overcome potential limitations of a current in vivo system including: 1) the cellular uptake of less-soluble unnatural amino acids and 2) the concentration of tRNA charged with the unnatural amino acid. Using this system we have incorporated unnatural amino acids with high selectivity at yields an order of magnitude higher than in vivo systems. By incorporating unnatural amino acids with either a terminal-azide or terminal-alkyne side chain we have used the biocompatible copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition to site-specifically conjugate two heterologous proteins. We have demonstrated (for the first time to our knowledge) a one-step, site-specific, direct protein-to-protein conjugation technique that does not require a chemical linker. This technique enables protein engineering for applications such as: 1) linking protein complexes together to from protein-based materials, 2) decorating protein complexes such as virus-like particles for vaccine and drug delivery applications, and 3) creating fusion proteins when a single polypeptide of the fusion partners does not express and/or fold correctly.