title: Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.
abstract: To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan. m-muraki@aist.go.jp
authors.forename: Michiro
authors.initials: M
authors.lastname: Muraki
authors.forename: Shinya
authors.initials: S
authors.lastname: Honda
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D019014:fas Receptor / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D013917:Thrombin