BACKGROUND: Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. RESULTS: Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. CONCLUSION: A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.
Reulen, S. W. A., van Baal, I., Raats, J. M. H., & Merkx, M. (2009). Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester. BMC Biotechnology, 9, 66. https://doi.org/10.1186/1472-6750-9-66