Protein bioconjugation sites

Gaertner, H.F., and Offord, R.E. (1996) Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins. Bioconjugate. Chem. 7, 38-44. 

Ni, J., Singh, S., Wang, L. X. (2003). Synthesis of maleimide-activated carbohydrates as chemoselective tags for site-specific glycosylation of peptides and proteins. Bioconjug. Chem., 14, 232-238. 

Fig. 10.3-2 Common strategies for protein bioconjugation, targeting lysine, cysteine, aspartic acid, and glutamic acid residues. In most situations, only cysteine modification reactions are site selective.

Several reports have been made using this reaction as a protein bioconjugation technique. In the first report, 60 azide functional groups attached to the surface of the cowpea mosaic virus (CPMV) using NHS ester or iodoacetamide reactions served as attachment sites for fluorescent alkyne derivatives [92]. The optimal conditions for the reaction were 21 pM protein (based on azide-functionalized capsid monomers), 1 mM CuS04, 2 mM TCEP, and 2 mM 58 in pH 8 buffer at 4°C for 16 h. As an alternative, small amounts of copper wire were also effective in generating and maintaining the Cu(I) species. The reaction was also successful when the alkynes were attached to the viral capsid and azides were attached to the dye. 

This method, while effective in creating bioconjugates, does not produce the well-defined conjugates required for some applications. Because each polymer contains multiple protein-reactive sites, each chain can bear a different number of proteins, thus creating a heterogeneous mixture of bioconjugates. End-group functionalized polymers circumvent this issue because they have either one or two protein-reaaive sites. 

ChiUcoti A, Chen G, Stayton PS, Hofftnan AS. Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein. Bioconjug. Chem. 1994 5 504-507. 

Geoghegan, K.E, and Stroh, J.G. (1992) Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Applications to modification at N-terminal serine. Bioconjugate Chem. 3, 138-146. 

Trubetskoy, V.S., Narula, J., Khaw, B.A., and Torchilin, V.P. (1993) Chemically optimized antimyosin Fab conjugates with chelating polymers Importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization. Bioconjugate Chem. 4, 251-255. 

Varenne, A., Salmain, M., Brisson, C., and Jaouen, G. (1992) Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy. Bioconjugate Chem. 3, 471-476. 

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