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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Autores principales:	Yosuke Hashimoto, Wei Zhou, Kohtaroh Hamauchi, Keisuke Shirakura, Takefumi Doi, Kiyohito Yagi, Tatsuya Sawasaki, Yoshiaki Okada, Masuo Kondoh, Hiroyuki Takeda
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8dea233c0348444988ed6f1f7cd39b73
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spelling	oai:doaj.org-article:8dea233c0348444988ed6f1f7cd39b732021-12-02T15:07:59ZEngineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-510.1038/s41598-018-26560-92045-2322https://doaj.org/article/8dea233c0348444988ed6f1f7cd39b732018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-26560-9https://doaj.org/toc/2045-2322Abstract 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.Yosuke HashimotoWei ZhouKohtaroh HamauchiKeisuke ShirakuraTakefumi DoiKiyohito YagiTatsuya SawasakiYoshiaki OkadaMasuo KondohHiroyuki TakedaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-12 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
author	Yosuke Hashimoto Wei Zhou Kohtaroh Hamauchi Keisuke Shirakura Takefumi Doi Kiyohito Yagi Tatsuya Sawasaki Yoshiaki Okada Masuo Kondoh Hiroyuki Takeda
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 Portfolio
publishDate	2018
url	https://doaj.org/article/8dea233c0348444988ed6f1f7cd39b73
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Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5

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