Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms

Abstract Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm...

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Autores principales:	Miyuki Kumano-Kuramochi, Ken-ichiro Tatematsu, Mayumi Ohnishi-Kameyama, Mari Maeda-Yamamoto, Toshiro Kobori, Hideki Sezutsu, Sachiko Machida
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fdc5dbf9aea54c0095b4790645242b43
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spelling	oai:doaj.org-article:fdc5dbf9aea54c0095b4790645242b432021-12-02T12:32:07ZCo-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms10.1038/s41598-017-00420-42045-2322https://doaj.org/article/fdc5dbf9aea54c0095b4790645242b432017-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-00420-4https://doaj.org/toc/2045-2322Abstract Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10−7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.Miyuki Kumano-KuramochiKen-ichiro TatematsuMayumi Ohnishi-KameyamaMari Maeda-YamamotoToshiro KoboriHideki SezutsuSachiko MachidaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Miyuki Kumano-Kuramochi Ken-ichiro Tatematsu Mayumi Ohnishi-Kameyama Mari Maeda-Yamamoto Toshiro Kobori Hideki Sezutsu Sachiko Machida Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
description	Abstract Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10−7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.
format	article
author	Miyuki Kumano-Kuramochi Ken-ichiro Tatematsu Mayumi Ohnishi-Kameyama Mari Maeda-Yamamoto Toshiro Kobori Hideki Sezutsu Sachiko Machida
author_facet	Miyuki Kumano-Kuramochi Ken-ichiro Tatematsu Mayumi Ohnishi-Kameyama Mari Maeda-Yamamoto Toshiro Kobori Hideki Sezutsu Sachiko Machida
author_sort	Miyuki Kumano-Kuramochi
title	Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
title_short	Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
title_full	Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
title_fullStr	Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
title_full_unstemmed	Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms
title_sort	co-expression of bira with biotin bait achieves in vivo biotinylation of overexpressed stable n-glycosylated srage in transgenic silkworms
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/fdc5dbf9aea54c0095b4790645242b43
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Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms

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