IgY‐binding peptide screened from a random peptide library as a ligand for IgY purification
IgY‐binding peptide screened from a random peptide library as a ligand for IgY purification
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从随机肽库中筛选出的 IgY 结合肽作为 IgY 纯化的配体
作者：
Kamrul Hasan Khan;Arisa Himeno;Shouhei Kosugi;Y. Nakashima;Abdur Rafique;Ayana Imamura;T. Hatanaka;D. Kato;Yuji Ito
鸡卵黄免疫球蛋白（IgY）是一种功能性的哺乳动物IgG的替代品，用于抗原检测。传统的IgY纯化方法涉及多步程序，导致IgY的纯度和回收率低。在这项研究中，我们开发了一个简单的IgY纯化系统，使用IgY特异性肽通过T7噬菌体展示技术鉴定。从在T7噬菌体上构建的二硫键限制的随机肽文库中，我们通过重复的生物淘选鉴定了三个特异性结合克隆（Y 4 - 4、Y 5 - 14和Y 5 - 55）。经表面等离子体共振分析，合成肽与IgY-Fc的结合特异性高，亲和力中等（Kd：Y 4 - 4 = 7.3 ± 0.2 μM，Y 5 - 55 = 4.4 ± 0.1 μM）。为了评估纯化IgY的能力，我们使用IgY结合肽进行免疫沉淀和亲和高效液相色谱;结果表明这些肽可以用作IgY纯化的亲和配体。然后，我们使用肽缀合柱从使用优化的脱脂技术预处理的蛋黄中纯化IgY。在这里，我们报告了一个具有成本效益的一步IgY纯化系统的构建，具有高纯度和回收率。版权所有© 2017作者. Journal of Peptide Science由European Peptide Society和John Wiley & Sons Ltd出版。
Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi‐step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY‐specific peptides identified by T7 phage display technology. From disulfide‐constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4‐4, Y5‐14, and Y5‐55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY‐Fc and moderate affinity for IgY‐Fc (Kd: Y4‐4 = 7.3 ± 0.2 μM and Y5‐55 = 4.4 ± 0.1 μM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high‐performance liquid chromatography using IgY‐binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide‐conjugated column to purify IgY from egg yolks pre‐treated using an optimized delipidation technique. Here, we report the construction of a cost‐effective, one‐step IgY purification system, with high purity and recovery. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.