Site-specific modification of a single-chain antibody using a novel glyoxylyl-based labeling reagent

A novel, highly specific protein modification approach is described. By usi ng conventional molecular cloning techniques, a protein can be constructed and expressed such that the N-terminal residue is replaced by cysteine. Its 1,2-aminothiol structure reacts very specifically with a glyoxylyl group a t pH 7 or below, forming a relatively stable thiazolidine bridge. Therefore , a glyoxylyl-based labeling agent (e.g., radioactive tags, fluorescent pro bes, biotin) can be used to specifically modify a protein at its N-terminus . To highlight this novel approach, a recombinant anti-insulin single chain antibody (scFv) was specifically biotinylated at its N-terminus even in th e presence of other proteins in the total cell lysate. The glyoxylyl-biotin ylated scFv retained binding activity similar to unmodified scFv.