title: Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.
abstract: The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.
authors.affiliation: Laboratory of Vaccinology and Vaccine Immunology, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tamaki
authors.affiliation: Laboratory of Vaccinology and Vaccine Immunology, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Harakuni
authors.affiliation: Laboratory of Vaccinology and Vaccine Immunology, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Rui
authors.initials: R
authors.lastname: Yamaguchi
authors.affiliation: Laboratory of Vaccinology and Vaccine Immunology, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyata
authors.affiliation: Laboratory of Vaccinology and Vaccine Immunology, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan. Electronic address: tarakawa@comb.u-ryukyu.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Arakawa
mesh_terms: D000818:Animals; D002772:Cholera Toxin / Q000737:chemistry*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D002479:Inclusion Bodies / Q000737:chemistry*; D008807:Mice, Inbred BALB C; D017510:Protein Folding; D021382:Protein Sorting Signals; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002772:Cholera Toxin
keywords: Cholera toxin B subunit; Dialysis; Multisubunit protein refolding; Pentamer