title: Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.
abstract: To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.
authors.affiliation: Molecular Microbiology Group, Department of Infectious Diseases, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Okinawa, Japan. Electronic address: tarakawa@comb.u-ryukyu.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Arakawa
authors.affiliation: Molecular Microbiology Group, Department of Infectious Diseases, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Okinawa, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Harakuni
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D064236:Cartilage Oligomeric Matrix Protein / Q000737:chemistry*; D002772:Cholera Toxin / Q000737:chemistry* / Q000276:immunology; D005260:Female; D007074:Immunoglobulin G / Q000097:blood; D008807:Mice, Inbred BALB C; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D064236:Cartilage Oligomeric Matrix Protein; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin
keywords: Cartilage oligomeric matrix protein; Cholera toxin B subunit; Physicochemical stability; Rational design of vaccines; α-Helical coiled coil