Chapter 12: Site-specific Protein Modification and Bio-orthogonal Chemistry

The modification of expressed proteins is critical to numerous applications in biochemistry and cell biology, including for fluorescence labelling for imaging and biophysics, for immobilization and affinity purification and for conjugation of small molecules and solubilizing groups. Strategies for protein modification can be broadly divided into categories based on the generality of the approach: methods which are specific for particular naturally-occurring amino acids, methods which require a specific sequence of amino acids and methods which require metabolic incorporation of unnatural, biorthogonal functional groups either in place of natural amino acids or as part of post-translational modifications. In all three cases, modern approaches to protein modification frequently rely upon the use of bio-orthogonal reactions in which functional groups with reactivity orthogonal to that found in natural biomolecules is used to direct protein modification.