Site-specific, bioorthogonal protein labeling using Endogenous Β-Amino Acid Dienophiles derived from natural product biosynthesis Daniel Richter, Edgars Lakis, Jörn Piel ETH Zurich, Switzerland Bioorthogonal chemistry has found widespread use in research and medicine by bringing rare chemical transformations into the diverse and crowded environment of living cells. Site-specific modification of proteins is of great interest for research applications and the preparation of clinically relevant protein conjugates. Intracellular labeling enables tracking of proteins involved in disease and subsequent modification to cause physiological changes. However, bioorthogonal reactions pose a few challenging requirements to be of use. Reactions need to proceed selectively, fast, and in aqueous environments. Tetrazine ligations are commonly used for bioorthogonal modifications due to their versatility, site-specificity, and fast reaction kinetics. Unnatural amino acids are installed in proteins by amber codon suppression or enzymatic ligations. These approaches can suffer from low yields, metabolic changes and interferences in natural pathways which is why novel approaches are needed. We herein report autonomous dienophile generation in bacteria termed TyrEx ( Tyramine Ex cision). We leveraged an enzymatic reaction naturally occurring in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) for use in bioorthogonal labeling of proteins. By utilizing an enzymatic post-translational modification (protein splicing), we have incorporated an aminopyruvate residue in ribosomally produced proteins. [2,3]This building block does not occur naturally in common model organisms and reacts with commercially available tetrazine derivatives in a fast and selective manner. We have characterized and studied this reaction and have shown that the reaction proceeds in aqueous media and intracellularly in Escherichia coli (E. coli) . We have demonstrated the utility of this method by producing and purifying a Her2-binding Affibody, which we characterized to be fully functional after conjugation. Additionally, we have labeled the bacterial cell division protein FtsZ with a fluorescent dye to investigate the localization of the protein in E. coli. We anticipate that the method will be useful for future in vivo studies of cellular proteins and as a new method for the engineering of protein conjugates References 1. Richter, D., Lakis, E., Piel, J. Site-Specific, Bioorthogonal Protein Labeling by Tetrazine Ligation using Endogenous β-Amino Acid Dienophiles. In preparation .Morinaka, B. I. et al. Natural Noncanonical Protein Splicing Yields Products with Diverse β-Amino Acid Residues. Science 359 , 779-782, (2018). 2. E. Lakis, M. S., J. Piel. In-Vivo Production of Diverse β-Amino Acid-Containing Proteins. Submitted , (2022).
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