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

• Published in: Scientific reports 2017-03, Vol.7 (1), p.356-356, Article 356
• Main Authors: Kumano-Kuramochi, Miyuki, Tatematsu, Ken-ichiro, Ohnishi-Kameyama, Mayumi, Maeda-Yamamoto, Mari, Kobori, Toshiro, Sezutsu, Hideki, Machida, Sachiko
• Format: Article
• Language: English
• Subjects: 631/45/611; 631/61/185; 82/103; 82/16; 82/58; 82/80; 82/83; Humanities and Social Sciences; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-00420-4

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.