Site-Specific Modification of Single-Chain Affinity Ligands for Fluorescence Labeling, Radiolabeling, and Bioconjugation

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2021 Aug 13

PMID 34386959

Citations 3

Authors

Boya Zhang

Sachith M Vidanapathirana

Colin F Greineder

Affiliations

Soon will be listed here.

Abstract

Single-chain protein affinity ligands are recombinant polypeptides that recreate the antigen-binding site of parental, monoclonal antibodies (mAbs) or present unique binding surfaces derived from display technologies, computational design, or other approaches. These diverse ligands have several advantages over full-length mAbs as agents for delivery of small molecule, protein, and nanoparticle cargoes to desired sites in the body. However, they present unique challenges for modification and bioconjugation. Fusion of a LPXTGG motif, or "sortag," and a 5-amino acid, flexible linker to the C-terminus of these affinity ligands enables high-efficiency transpeptidation by the bacterial enzyme, Sortase A, and site-specific addition of fluorophores, radiolabels, or functional groups for oriented and stoichiometrically controlled bioconjugation. We describe in detail this method and address several challenges and pitfalls in the purification and characterization of modified single-chain affinity ligands.

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