Production of Rous sarcoma virus-like particles displaying human transmembrane protein in silkworm larvae and its application to ligand-receptor binding assay

摘要

Two types of Rous sarcoma virus (RSV) group-antigen protein (Gag) virus like particles (VLPs), full-length Gag (Gag701) and RSV protease domain (PR)-deleted mutant (Gag577) were expressed in silkworm larvae. Gag577 was secreted into hemolymph efficiently using wild type bacmid (WT), cysteine protease-deficient bacmid (CP-), cysteine protease and chitinase-deficient bacmid (CP(-)Chi(-)) bacmids, but comparatively Gag701 secretion levels were low. VLPs were purified on 10-60% (v/v) sucrose density gradient by ultracentrifugation and their structures confirmed under electron microscope. When hPRR and RSV Gag577 were co-expressed in silkworm larvae, human prorenin receptor (hPRR) was displayed on the surface of RSV VLPs, which was detected by Western blotting and immunoelectron microscopy. Moreover, binding of hPRR localized on the surface of VLPs to human prorenin was confirmed by ELISA. These results indicate that active hPRR was displayed on the surface of RSV VLPs, which can be utilized for drug discovery of hPRR blockers to prevent nephropathy. Moreover, this transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assay of transmembrane proteins to its ligands, especially for transmembrane proteins which cannot be purified from membrane fractions in active states