Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein-protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-L-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-L-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the singlestep Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0] non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.

Automated summary

Genetic code engineering is used to modify proteins for enhanced biological availability, with the addition of 3,4-dihydroxy-L-phenylalanine through tyrosinase catalysis. This method allows for selective cell labeling and improved sensitivity for protein-based fluorescence labeling.