Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5.
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5.
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DOI：
10.1038/s41598-018-26560-9
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发表时间：
2018-05-30
影响因子：
4.6
通讯作者：
Takeda H
中科院分区：
文献类型：
--
作者：
Hashimoto Y;Zhou W;Hamauchi K;Shirakura K;Doi T;Yagi K;Sawasaki T;Okada Y;Kondoh M;Takeda H
众所周知，针对多跨膜蛋白胞外区（ECR）的抗体的生产非常困难，因为膜蛋白由于其复杂的结构和物种间高度保守的序列而导致生产率低和免疫原性低。在这里，我们介绍了一种利用小麦无细胞蛋白质合成系统制备的工程脂质体免疫原生成 ECR 结合抗体的新方法。我们使用claudin-5 (CLDN-5)作为靶抗原，这是一种众所周知难以生产且免疫原性差的膜蛋白，具有两个高度保守的细胞外环。在抑制和标准化 mRNA GC 含量后，我们极大地提高了无细胞系统中 CLDN-5 的生产力。为了克服其低免疫原性，设计并合成了两种工程抗原作为脂蛋白体：人/小鼠嵌合 CLDN-5 和由对称排列的 ECR 组成的基于 CLDN-5 的人工膜蛋白。两种工程化 CLDN-5 ECR 抗原的腹膜内免疫可在小鼠体内诱导 ECR 结合抗体，成功率很高。我们分离了五种特异性识别 CLDN-5 ECR 的单克隆抗体。抗体克隆 2B12 显示出高亲和力 (<10nM) 并抑制含有 CLDN-5 的紧密连接。这些结果证明了针对难以生产的膜蛋白（例如 CLDN）的单克隆抗体开发方法的有效性。
The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.
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影响因子：
4.6
作者：
Takeda H;Ogasawara T;Ozawa T;Muraguchi A;Jih PJ;Morishita R;Uchigashima M;Watanabe M;Fujimoto T;Iwasaki T;Endo Y;Sawasaki T
通讯作者：
Sawasaki T
影响因子：
5.8
作者：
Webb, David R.;Handel, Tracy M.;Kretz-Rommel, Anke;Stevens, Raymond C.
通讯作者：
Stevens, Raymond C.
影响因子：
7.8
作者：
Nitta, T;Hata, M;Tsukita, S
通讯作者：
Tsukita, S
影响因子：
3.5
作者：
Nozawa A;Ogasawara T;Matsunaga S;Iwasaki T;Sawasaki T;Endo Y
通讯作者：
Endo Y
影响因子：
3.7
作者：
Yano T;Takeda H;Uematsu A;Yamanaka S;Nomura S;Nemoto K;Iwasaki T;Takahashi H;Sawasaki T
通讯作者：
Sawasaki T