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.
Highlights
The receptor for advanced glycation end products (RAGE) was originally identified as a receptor for advanced glycation end products (AGEs) and has been shown to be involved in the pathogenesis of diabetic complications
No signal was detected in the negative control, whereas strong signals were detected in all lines in which the expression of sRAGE in middle silk glands (MSGs) was under the control of the GAL4/UAS system
These data suggested that the GAL4/UAS system could be used for expression of human sRAGE in MSGs of transgenic silkworms
Summary
The receptor for advanced glycation end products (RAGE) was originally identified as a receptor for advanced glycation end products (AGEs) and has been shown to be involved in the pathogenesis of diabetic complications. Recent studies have shown that RAGE is expressed in multiple cell types and functions as a pattern recognition receptor, binding structurally diverse ligands, such as amyloid-β, members of the S100 protein superfamily, and high-mobility group box-1 protein (HMGB-1)[3,4,5]. SRAGE has been expressed and purified from HEK293, Escherichia coli, Baculovirus, and yeast Pichia pastoris expression systems[2, 18, 19] These expression systems do not yield sufficient amounts of sRAGE, mostly due to folding problems, protein instability, and high cost. Our results demonstrated the production of N-glycosylated, biotinylation domain-tagged sRAGE in MSGs of transgenic silkworms, facilitating biotinylation after co-expression of biotin ligase (BirA) and addition of biotin bait
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