Inducible, Selective Labeling of Proteins via Enzymatic Oxidation of Tyrosine.

Proteins can be labeled site-specifically and in inducible fashion by exposing a small peptide tag (G4Y) on any of its termini and activating the newly exposed tyrosine residue with the enzyme mushroom tyrosinase. The enzyme generates a quinone by oxidizing the tyrosine, which in turn can perform strain-promoted oxidation-controlled ortho-quinone cycloaddition (SPOCQ) with strained alkynes and alkenes, generating a stable conjugation product. Here, we describe a protocol to perform SPOCQ reaction on proteins, along with notes to optimize yield and reaction rates. Conjugation efficiencies of over 95% to antibodies have been reported using this protocol.

Highly Efficient Mono-Functionalization of Knob-in-Hole Antibodies with Strain-Promoted Click Chemistry.

Knob-in-hole antibodies can be utilized to introduce a single tag for chemo-enzymatic functionalization. By either introducing a single C-terminal sortase tag (sortase-tag expressed protein ligation) or tyrosine tag (G4Y), mono-functionalization of the monoclonal antibody trastuzumab was achieved rapidly and in high yields. This method was applied to selectively and efficiently introduce a single fluorescent tag, cytokine or single-chain variable fragment, as well as produce clean homo dimers of trastuzumab.