Sortase-Tag Expressed Protein Ligation: Combining
Protein Purification and Site-Specific Bioconjugation into a Single
Step
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Abstract
Efficient labeling of protein-based
targeting ligands with various
cargos (drugs, imaging agents, nanoparticles, etc.) is essential to
the fields of molecular imaging and targeted therapeutics. Many common
bioconjugation techniques, however, are inefficient, nonstoichiometric,
not site-specific, and/or incompatible with certain classes of protein
scaffolds. Additionally, these techniques can result in a mixture
of conjugated and unconjugated products, which are often difficult
to separate. In this study, a bacterial sortase enzyme was utilized
to condense targeting ligand purification and site-specific conjugation
at the C-terminus into a single step. A model was produced to determine
optimal reaction conditions for high conjugate purity and efficient
utilization of cargo. As proof-of-principle, the sortase-tag expressed
protein ligation (STEPL) technique was used to generate tumor-specific
affinity ligands with fluorescent labels and/or azide modifications
at high purity (>95%) such that it was not necessary to remove
unconjugated
impurities. Click chemistry was then used for the highly efficient
and site-specific attachment of the azide-modified targeting ligands
onto nanoparticles