(188bs) Site-Specific Conjugation of Scfv Using the Nucleotide Binding Site
However, several challenges in terms of conjugation and purification limit the potential of these molecules in academia and potential commercialization. Common conjugation procedures rely on the reactive side chains of surface-exposed amino acids such as lysines. While simple and efficient, these processes are not site-specific, yielding heterogenous products and potentially affecting the binding activity of the scFv. Alternatively, complex protein engineering methods for addition of conjugation sites can be used on a case-by-case basis, but present challenges in maintaining proper folding and binding activity once the scFv has been modified.
One potential solution to these problems is the use of the Nucleotide Binding Site (NBS), initially identified as a highly conserved region in the variable domain of Fab region in antibodies and conserved in scFv. Using a UV-mediated photocrosslinking mechanism (UV-NBS) and previously identified ligands with moderate affinity for the NBS, typically small ring structured molecules, we evaluate the covalent site-specific conjugation of functional moieties to scFv. Thanks to the conservation of the NBS, this presents a viable process for conjugation of most scFv. We optimize the conditions required for efficient conjugation including incubation times, UV exposure and ligand concentration for two different functionalities - biotin and fluorescein. Importantly, we assess conservation of the binding activity in final products, accessibility of the conjugated functionality and conjugation yields. Overall, this process represents an attractive alternative for the reproducible production of active, homogeneously labeled scFv for use in downstream diagnostic and therapeutic applications.