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Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.
- Source :
-
Scientific reports [Sci Rep] 2017 Mar 23; Vol. 7 (1), pp. 356. Date of Electronic Publication: 2017 Mar 23. - Publication Year :
- 2017
-
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 1âmg 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 7.25â×â10 <superscript>-7</superscript> âM, 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.
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 7
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 28336960
- Full Text :
- https://doi.org/10.1038/s41598-017-00420-4