Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5
Abstract 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 intr...
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doaj-8dea233c0348444988ed6f1f7cd39b732020-12-08T04:45:09ZengNature Publishing GroupScientific Reports2045-23222018-05-018111210.1038/s41598-018-26560-9Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5Yosuke Hashimoto0Wei Zhou1Kohtaroh Hamauchi2Keisuke Shirakura3Takefumi Doi4Kiyohito Yagi5Tatsuya Sawasaki6Yoshiaki Okada7Masuo Kondoh8Hiroyuki Takeda9Graduate School of Pharmaceutical Sciences, Osaka UniversityProteo-Science Center, Ehime UniversityProteo-Science Center, Ehime UniversityGraduate School of Pharmaceutical Sciences, Osaka UniversityGraduate School of Pharmaceutical Sciences, Osaka UniversityGraduate School of Pharmaceutical Sciences, Osaka UniversityProteo-Science Center, Ehime UniversityGraduate School of Pharmaceutical Sciences, Osaka UniversityGraduate School of Pharmaceutical Sciences, Osaka UniversityProteo-Science Center, Ehime UniversityAbstract 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.https://doi.org/10.1038/s41598-018-26560-9 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yosuke Hashimoto Wei Zhou Kohtaroh Hamauchi Keisuke Shirakura Takefumi Doi Kiyohito Yagi Tatsuya Sawasaki Yoshiaki Okada Masuo Kondoh Hiroyuki Takeda |
spellingShingle |
Yosuke Hashimoto Wei Zhou Kohtaroh Hamauchi Keisuke Shirakura Takefumi Doi Kiyohito Yagi Tatsuya Sawasaki Yoshiaki Okada Masuo Kondoh Hiroyuki Takeda Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 Scientific Reports |
author_facet |
Yosuke Hashimoto Wei Zhou Kohtaroh Hamauchi Keisuke Shirakura Takefumi Doi Kiyohito Yagi Tatsuya Sawasaki Yoshiaki Okada Masuo Kondoh Hiroyuki Takeda |
author_sort |
Yosuke Hashimoto |
title |
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
title_short |
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
title_full |
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
title_fullStr |
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
title_full_unstemmed |
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
title_sort |
engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2018-05-01 |
description |
Abstract 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. |
url |
https://doi.org/10.1038/s41598-018-26560-9 |
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