SELECTION OF ANTIBODY LIGANDS FROM A LARGE LIBRARY OF OLIGOPEPTIDES EXPRESSED ON A MULTIVALENT EXPOSITION VECTOR
SELECTION OF ANTIBODY LIGANDS FROM A LARGE LIBRARY OF OLIGOPEPTIDES EXPRESSED ON A MULTIVALENT EXPOSITION VECTOR
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DOI：
10.1016/0022-2836(91)90213-p
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发表时间：
1991-11-20
影响因子：
5.6
通讯作者：
CESARENI, G
中科院分区：
文献类型：
--
作者：
FELICI, F;CASTAGNOLI, L;CESARENI, G
实际上，任何寡肽都可以通过与丝状噬菌体的主要外壳蛋白融合而暴露在噬菌体衣壳的表面上。表达特定肽标签的噬菌体可以通过用蛋白质配体进行亲和纯化，从暴露随机序列的寡肽的数千万个克隆的混合物中选择。在这方面，pVIII可用作基于基因III产物（pIII）的暴露载体的替代和补充。我们已经构建了一个噬菌粒载体，含有基因VIII的pLac启动子的控制下。该载体可方便地用于构建具有随机氨基酸序列的寡肽文库。使用抗肽单克隆抗体亲和纯化噬菌粒，暴露可与单克隆抗体相互作用的寡肽。回收的克隆的基因VIII的氨基末端的DNA测序预测杂合蛋白的合成，所述杂合蛋白的氨基末端氨基酸序列与用于产生抗体的寡肽的氨基末端氨基酸序列相关。换句话说，只有结合免疫球蛋白G表面非常小部分的寡肽通过该方法亲和纯化，这意味着抗原结合位点具有使其比分子的其余部分更粘的分子性质。
Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose amino-terminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.