Site-specific conjugation of fibroblast growth factor 2 (FGF2) based on incorporation of alkyne-reactive unnatural amino acid.

Recent advances in site-specific protein modification include the increasingly popular incorporation of unnatural amino acid(s) using amber codon, a method developed by Schultz and coworkers. In this study, we employ this technique to introduce propargyllysine (PrK) in human fibroblast growth factor 2 (FGF2). Owing to an alkyne moiety in its side chain, PrK is compatible with Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). We successfully tested CuAAC-mediated conjugation of FGF2 with two compounds - a fluorophore carboxyrhodamine 110 or a cytotoxic drug monomethyl auristatin E (MMAE). In the case of the MMAE conjugate we improved the initial poor conjugation yield to achieve nearly 100% efficiency after extensive optimization. The detergent-based optimization approach may help overcome problems with the CuAAC reaction yield for protein modification with hydrophobic compounds, such as MMAE.