Sybodies (Synthetic single domain antibodies) are small single-domain antibodies, the structure of which is based on camelid heavy-chain-only antibody binding domains (Nanobodies). Sybodies that bind against specific proteins can be selected using only in vitro methods. High levels of Cathepsin B (KatB) are found in a wide variety of cancers, making it a promising target for the development of molecular probes for non-invasive imaging. Due to their small size sybody-based molecular probes would have rapid clearance and therefore low background signal. Here we labeled »Sy7«, a sybody targeting human and mouse KatB, with a near-infrared (NIR) fluorescent dye Cyanine 5.5 (Cy5.5). First, we labeled a CLPETGY peptide with Cy5.5 using a maleimide reaction. We then attached the labeled peptide Cy5.5CLPETGY to the N-terminus of a modified Sy7 sybody by sortase A-mediated reaction. We found that the labeled and purified Sy7GGCy5.5 still retains the ability to bind to KatB. Alas using the methods we employed, the production of greater amounts of Sy7GGCy5.5 is limited due to the low yield and long duration of some steps. Our methods, therefore, require further optimization.
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