Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast

• Published in: Scientific reports Vol. 5; no. 1; p. 7678
• Main Authors: Liu, Bo, Shi, DanYang, Chang, ShaoHong, Gong, Xin, Yu, YunZhou, Sun, ZhiWei, Wu, Jun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08-01-2015; Nature Publishing Group
• Subjects: 38/39; 38/44; 631/250/590/2294; 631/61/185; 82/80; 82/83; Animals; Antibodies, Neutralizing - immunology; Antigens; Botulinum toxin; Botulinum toxin type B; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - immunology; Botulinum Toxins, Type A - metabolism; Botulism; Clostridium botulinum - metabolism; Deglycosylation; Enzyme-Linked Immunosorbent Assay; Female; Food contamination; Glycosylation; Humanities and Social Sciences; Immunology; Mice; Mice, Inbred BALB C; multidisciplinary; Mutagenesis, Site-Directed; Neurotoxins; Peptides; Protein Sorting Signals - genetics; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae - metabolism; Science; Serogroup; Tetanus; Tetanus toxin; Toxins; Vaccines, Subunit - immunology; Yeast; Yeasts
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep07678

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.