title: Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity.
abstract: Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Sung In
authors.initials: SI
authors.lastname: Lim
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Aichi, Japan.
authors.forename: Yukina
authors.initials: Y
authors.lastname: Mizuta
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Aichi, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Takasu
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, Seoul, Republic of Korea.
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, United States of America; School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001483:Base Sequence; D003300:Copper / Q000737:chemistry*; D061565:Cycloaddition Reaction; D017931:DNA Primers; D051379:Mice; D016133:Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
chemical_list: D000480:Alkynes; D001386:Azides; D017931:DNA Primers; D003300:Copper; D013762:Tetrahydrofolate Dehydrogenase