Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity
Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa ß2-microglobulin (ß2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class...
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Language: | English |
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ftunivwagenin:oai:library.wur.nl:wurpubs/372046 2024-02-04T09:58:57+01:00 Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity Zhao, H. Stet, R.J.M. Skjodt, K. Savelkoul, H.F.J. 2008 application/pdf https://research.wur.nl/en/publications/expression-and-characterization-of-recombinant-single-chain-salmo https://doi.org/10.1016/j.fsi.2008.01.003 en eng https://edepot.wur.nl/29882 https://research.wur.nl/en/publications/expression-and-characterization-of-recombinant-single-chain-salmo doi:10.1016/j.fsi.2008.01.003 info:eu-repo/semantics/restrictedAccess Wageningen University & Research Fish and Shellfish Immunology 24 (2008) 4 ISSN: 1050-4648 anemia-virus atlantic salmon cell-surface histocompatibility class-i identification molecules peptides polymorphism salar l t-cells info:eu-repo/semantics/article Article/Letter to editor info:eu-repo/semantics/publishedVersion 2008 ftunivwagenin https://doi.org/10.1016/j.fsi.2008.01.003 2024-01-10T23:24:16Z Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa ß2-microglobulin (ß2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of ß2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasaß2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and ß2m, and showed binding to the recombinant molecule. The recombinant complex Sasaß2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against ß2m and ¿3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens. Article in Journal/Newspaper Atlantic salmon Wageningen UR (University & Research Centre): Digital Library Fish & Shellfish Immunology 24 4 459 466 |
institution |
Open Polar |
collection |
Wageningen UR (University & Research Centre): Digital Library |
op_collection_id |
ftunivwagenin |
language |
English |
topic |
anemia-virus atlantic salmon cell-surface histocompatibility class-i identification molecules peptides polymorphism salar l t-cells |
spellingShingle |
anemia-virus atlantic salmon cell-surface histocompatibility class-i identification molecules peptides polymorphism salar l t-cells Zhao, H. Stet, R.J.M. Skjodt, K. Savelkoul, H.F.J. Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
topic_facet |
anemia-virus atlantic salmon cell-surface histocompatibility class-i identification molecules peptides polymorphism salar l t-cells |
description |
Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa ß2-microglobulin (ß2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of ß2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasaß2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and ß2m, and showed binding to the recombinant molecule. The recombinant complex Sasaß2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against ß2m and ¿3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens. |
format |
Article in Journal/Newspaper |
author |
Zhao, H. Stet, R.J.M. Skjodt, K. Savelkoul, H.F.J. |
author_facet |
Zhao, H. Stet, R.J.M. Skjodt, K. Savelkoul, H.F.J. |
author_sort |
Zhao, H. |
title |
Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
title_short |
Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
title_full |
Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
title_fullStr |
Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
title_full_unstemmed |
Expression and characterization of recombinant single-chain salmon class I MHC fused with ß2-microglobulin with biological activity |
title_sort |
expression and characterization of recombinant single-chain salmon class i mhc fused with ß2-microglobulin with biological activity |
publishDate |
2008 |
url |
https://research.wur.nl/en/publications/expression-and-characterization-of-recombinant-single-chain-salmo https://doi.org/10.1016/j.fsi.2008.01.003 |
genre |
Atlantic salmon |
genre_facet |
Atlantic salmon |
op_source |
Fish and Shellfish Immunology 24 (2008) 4 ISSN: 1050-4648 |
op_relation |
https://edepot.wur.nl/29882 https://research.wur.nl/en/publications/expression-and-characterization-of-recombinant-single-chain-salmo doi:10.1016/j.fsi.2008.01.003 |
op_rights |
info:eu-repo/semantics/restrictedAccess Wageningen University & Research |
op_doi |
https://doi.org/10.1016/j.fsi.2008.01.003 |
container_title |
Fish & Shellfish Immunology |
container_volume |
24 |
container_issue |
4 |
container_start_page |
459 |
op_container_end_page |
466 |
_version_ |
1789963543580246016 |