Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

Protein modification is extremely useful for both understanding of protein structure and function and the mechanism of the biological pathways that the protein is involved. For example, protein labeling can facilitate protein localization, binding partner identification, and purification both under native or denaturing conditions.[1] Furthermore, modification of protein can expand the proteins’ functional capacity, especially, for therapeutic proteins, which is very essential for enhancing their pharmacodynamic and pharmacokinetic properties.[2] The key point for a practical protein modification is to carry out site-specific chemistry so as to avoid upsetting the protein activity due to random modification since there is a diversity of chemically reactive functionalities in the protein in general. Particularly, site-specific attachment of two or more different functionalities to a protein is even more challenging than introducing a single modification but is highly sought after.[3] For example, introducing an analytical probe at one site for protein tracking and a functional moiety at the other site for expanding its functional capacity offers an enormous way to generate therapeutic proteins with enhanced biological activity and stability while affording efficient biological activity evaluation both in vitro and in vivo as well.