AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.
AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.
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DOI：
10.1371/journal.pone.0156716
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Sawasaki T
中科院分区：
文献类型：
--
作者：
Yano T;Takeda H;Uematsu A;Yamanaka S;Nomura S;Nemoto K;Iwasaki T;Takahashi H;Sawasaki T
多肽标签技术广泛应用于蛋白质检测和亲和纯化。它由两个基本元件组成：肽序列和特异性结合肽标签的结合剂。在许多标签系统中，抗体由于其高亲和力和特异性而被用作结合剂。最近，我们获得了克隆Ra 48，一种高亲和力的抗多巴胺受体D1（DRD 1）的兔单克隆抗体（mAb）。在这里，我们报告了一种新的标签系统组成的Ra 48抗体及其表位序列。使用缺失测定，我们鉴定了DRD 1的C-末端区域中的EEAAGIARP作为Ra 48 mAb的最小表位，并且基于其中心序列，我们将该序列命名为“AGIA”标签。标签序列不包括对翻译后修饰敏感的四个氨基酸Ser、Thr、Tyr或Lys。我们证明了这种新的标签系统在生物化学和细胞生物学应用中的性能。SPR分析表明Ra 48 mAb与AGIA标签的亲和力为4.90 × 10−9 M。AGIA标签在免疫印迹中显示出非常高的灵敏度和特异性。用抗AGIA抗体进行免疫印迹和低背景免疫染色，可检测到大量在动物和植物细胞中过表达的AGIA融合蛋白，并能有效地进行免疫沉淀。此外，第二个Glu到Asp的单个氨基酸取代（AGIA/E2 D）使得能够通过添加野生型AGIA肽竞争性解离AGIA/E2 D标记的蛋白质。它能够在生理条件下通过肽竞争一步纯化AGIA/E2 D标记的重组蛋白。AGIA系统的灵敏度和特异性使其适合用于多种蛋白质分析方法。
Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.