Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.
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 × 10-7M, 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.
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Miyuki
M
Kumano-Kuramochi
Institute of Agrobiological Sciences, NARO, 1-2 Owashi, Tsukuba, Ibaraki, 305-8643, Japan.
Ken-Ichiro
KI
Tatematsu
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Mayumi
M
Ohnishi-Kameyama
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Mari
M
Maeda-Yamamoto
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Toshiro
T
Kobori
Institute of Agrobiological Sciences, NARO, 1-2 Owashi, Tsukuba, Ibaraki, 305-8643, Japan.
Hideki
H
Sezutsu
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan. lili@affrc.go.jp.
Sachiko
S
Machida
D000818:Animals; D030801:Animals, Genetically Modified; D001710:Biotin / Q000494:pharmacology*; D019921:Biotinylation; D012831:Bombyx / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D000067759:Receptor for Advanced Glycation End Products / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D001710:Biotin; D019731:Carbon-Nitrogen Ligases; D029968:Escherichia coli Proteins; D000067759:Receptor for Advanced Glycation End Products; D012097:Repressor Proteins; C058125:birA protein, E coli; C000593871:AGER protein, human