Site-Specific Modification of Single-Chain Affinity Ligands for Fluorescence Labeling, Radiolabeling, and Bioconjugation.
Site-Specific Modification of Single-Chain Affinity Ligands for Fluorescence Labeling, Radiolabeling, and Bioconjugation.
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DOI：
10.1007/978-1-0716-1617-8_15
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发表时间：
2021
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单链蛋白亲和配体是重组多肽，其重建亲本单克隆抗体（mAb）的抗原结合位点或呈现源自展示技术、计算设计或其他方法的独特结合表面。这些不同的配体具有超过全长mAb的几个优点，作为用于将小分子、蛋白质和纳米颗粒货物递送到体内所需部位的试剂。然而，它们对修饰和生物缀合提出了独特的挑战。LPXTGG基序或“分选标签”和5-氨基酸柔性接头与这些亲和配体的C-末端的融合使得能够通过细菌酶分选酶A进行高效转肽，并且位点特异性添加荧光团、放射性标记或官能团以用于定向和化学计量控制的生物缀合。我们详细描述了这种方法，并解决了几个挑战和陷阱的纯化和表征修饰的单链亲和配体。
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.