title: Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast.
abstract: The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens.
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: DanYang
authors.initials: D
authors.lastname: Shi
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: ShaoHong
authors.initials: S
authors.lastname: Chang
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: Xin
authors.initials: X
authors.lastname: Gong
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: YunZhou
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: ZhiWei
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003014:Clostridium botulinum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006031:Glycosylation; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D065288:Serogroup; D022223:Vaccines, Subunit / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; C096323:rimabotulinumtoxinB; D019274:Botulinum Toxins, Type A