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. 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.

Food functionality research as a new national project in special reference to improvement of cognitive and locomotive abilities

“…Platform IV (new functionality analysis), headed by Gen-Ichiro Soma (Control of Innate Immunity Technology Research Association), investigates bodily homeostasis for the assessment of food functionalities. A number of papers and patents have been produced [81][82][83][84][85][86][87][88][89][90][91][92][93][94][95]. To evaluate various aspects of the effect of AFF food products and sports on the bodily homeostasis, in addition to the usual biochemical testing, a basic system was constructed that enables using trace amounts (μl) of untreated blood samples for neutrophil, oxidized LDL, and phagocyte analyses.…”

“…The research group involved in this platform has also proposed an integrated protocol for the use of blood samples of animal and human origin. The most important accomplishments of the group thus far include (1) the first-ever patent pertaining to a construction of a device that can be used for simultaneous measurement of superoxide and myeloperoxidase activities [92,95]; (2) detection of oxidized LDL in human plasma using oxidized LDL receptor that is engineered in transgenic silkworms (detection limit: 10 ng/ml) [82,87,91,93]; and (3) construction of a firstever double-tube device for measuring phagocytosis [83,90,94] in the human peripheral blood. The members of the group are collaborating with platform I, II, and III group members to detect correlations between conventional biochemical parameters and these homeostasis monitoring factors ( Figure 5).…”