Screening of peptide ligands that bind to the Fc region of IgG using peptide array and its application to affinity purification of antibody
Screening of peptide ligands that bind to the Fc region of IgG using peptide array and its application to affinity purification of antibody
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DOI：
10.1016/j.bej.2013.06.017
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发表时间：
2013-10-15
影响因子：
3.9
通讯作者：
Honda, Hiroyuki
中科院分区：
文献类型：
--
作者：
Sugita, Tomoya;Katayama, Makoto;Honda, Hiroyuki
由于小肽与蛋白质配体相比更稳定、免疫原性更低且更便宜，因此其对于亲和纯化吸引了极大的兴趣。在本研究中，使用点合成的肽阵列，从IgG Fc γ受体的氨基酸序列研究了对IgG的Fc区具有亲和力的肽的筛选，所述IgG Fc γ受体是众所周知的IgG-Fc结合蛋白之一。通过氨基酸取代试验获得了对小鼠IgG具有高亲和力的八聚体肽NKFRGKYK和NARKFYKG。两种肽也识别人IgG-Fc。人IgG-Fc的结合常数为8.9 x 10（6）（NKFRGKYK）和6.5 x 10（6）M-1（NARKFYKG）。使用肽固定化树脂，来自细胞培养基的人IgG-Fc的纯度对于NKFRGKYK为83%，对于NARKFYKG为68%。这些肽配体用于从细胞培养上清液中纯化单克隆抗体（MAb）。发现对于NKFRGKYK和NARKFYKG，抗体获得的产率分别为69%和80%。通过这些肽树脂获得的残留DNA和宿主细胞蛋白减少分别在4.2和1.3对数减少值（LRV）（NKFRGKYK）、3.9和1.5 LRV（NARKFYKG）范围内，与蛋白A报告的结果相当。因此，表明筛选的NKFRGKYK和NARKFYKG肽可用作IgG的亲和纯化配体。(C)2013爱思唯尔有限公司版权所有。
Small peptides have attracted great interest for affinity purification since they are more stable, less immunogenic, and less expensive compared to protein ligands. In this study, screening of peptides that has affinity to Fc region of IgG was investigated from amino acid sequence of the IgG Fey receptor, which is well known as one of the IgG-Fc binding proteins, using a spot-synthesized peptide array. High affinity octamer peptides, NKFRGKYK and NARKFYKG, for mouse IgG were obtained by amino acid substitution assays. Both peptides also recognized human IgG-Fc. The association constants for human IgG-Fc were 8.9 x 10(6) (NKFRGKYK) and 6.5 x 10(6) M-1 (NARKFYKG). Purity of the human IgG-Fc from cell culture medium using the peptide immobilized resins was 83% for NKFRGKYK and 68% for NARKFYKG. These peptide ligands were used for the purification of monoclonal antibody (MAb) from cell culture supernatants. The yields obtained for the antibody were found to be 69% and 80% for NKFRGKYK and NARKFYKG, respectively. The residual DNA and host cell protein reduction obtained by these peptides resin were in the range of 4.2 and 1.3 log reduction value (LRV) (NKFRGKYK), 3.9 and 1.5LRV (NARKFYKG), respectively, comparable to those reported for Protein A. Therefore, it was indicated that the screened NKFRGKYK and NARKFYKG peptide would be useful as affinity purification ligands for IgG. (C) 2013 Elsevier B.V. All rights reserved.