Utilizing a Simple Method for Stoichiometric Protein Labeling to Quantify Antibody Blockade
Utilizing a Simple Method for Stoichiometric Protein Labeling to Quantify Antibody Blockade
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DOI：
10.1038/s41598-019-43469-z
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发表时间：
2019-05-07
影响因子：
4.6
通讯作者：
Wood, Keith, V
中科院分区：
文献类型：
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作者：
Ohana, Rachel Friedman;Hurst, Robin;Wood, Keith, V
配体结合测定通常使用荧光标记的蛋白质配体来量化结合程度。这些配体通常是通过对可接近的赖氨酸残基进行化学修饰而产生的，这通常会导致异质群体表现出可变的结合特性。这可以通过定量的、特定位点的标记来补救。最近，我们报道了一种将重组蛋白纯化与 2-氰基苯并噻唑 (CBT) 缩合相结合的单步方法，用于标记蛋白水解暴露的 N 末端半胱氨酸。在这里，我们使用三种生长因子表明，与随机赖氨酸标记不同，这种位点特异性方法产生了同质的生长因子群体，这些生长因子在其 N 末端进行定量标记并保留其结合特征。我们通过开发一种新的测定方法来证明这种标记方法的实用性，该测定方法可以量化抗体阻断受体-配体相互作用（即抗体阻断）的能力。该测定使用生物发光共振能量转移 (BRET) 来检测 CBT 标记的生长因子与其基因融合到 NanoLuc 荧光素酶的同源受体的结合。抗体阻断这些相互作用的能力通过 BRET 的减少来量化。使用多种抗体，我们证明该测定可以对细胞环境中的抗体阻断进行可靠的定量。正如这里所证明的，这种生成均匀标记蛋白质的简单方法有可能促进更准确和更稳健的配体结合测定。
Ligand binding assays routinely employ fluorescently-labeled protein ligands to quantify the extent of binding. These ligands are commonly generated through chemical modification of accessible lysine residues, which often results in heterogeneous populations exhibiting variable binding properties. This could be remedied by quantitative, site-specific labeling. Recently, we reported on a single-step method integrating recombinant protein purification with 2-cyanobenzothiazole (CBT) condensation for labeling a proteolytically exposed N-terminal cysteine. Here, using three growth factors, we show that unlike random lysine labeling, this site-specific approach yielded homogeneous populations of growth factors that were quantitatively labeled at their N-termini and retained their binding characteristics. We demonstrate the utility of this labeling method through the development of a novel assay that quantifies the capacity of antibodies to block receptor-ligand interactions (i.e. antibody blockade). The assay uses bioluminescence resonance energy transfer (BRET) to detect binding of CBT-labeled growth factors to their cognate receptors genetically fused to NanoLuc luciferase. The ability of antibodies to block these interactions is quantified through decrease in BRET. Using several antibodies, we show that the assay provides reliable quantification of antibody blockade in a cellular context. As demonstrated here, this simple method for generating uniformly-labeled proteins has potential to promote more accurate and robust ligand binding assays.