Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

摘要: Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking azido and alkynyl group in the presence of copper catalyst. Incorporation a non-natural amino acid (NAA) containing either or into protein allows site-specific bioconjugation mild conditions via CuAAC. Despite its great potential, enzyme has been hampered by several issues including low yield, poor solubility ligand, structural/functional perturbation CuAAC components. In present study, we incorporated alkyne-bearing NAA enzyme, murine dihydrofolate reductase (mDHFR), high cell density cultivation Escherichia coli, performed conjugation with fluorescent azide dyes to evaluate compatibility various comprising combination commercially available Cu(I)-chelating ligands reductants. The condensed culture improves yield 19-fold based on same amount acid, incubation under optimized condition did not lead any activity loss but allowed fast high-yield bioconjugation. Using established conditions, biotin-azide spacer was efficiently conjugated mDHFR retained leading immobilization biotin-conjugated streptavidin-coated plate. These results demonstrate that reactive incorporation can be used bioconjugate enzymes enzymatic activity.