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

Liu B, Shi D, Chang S, Gong X, Yu Y, Sun Z, Wu J - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of purified recombinant BHc products by SDS–PAGE.(A) Hyper-glycosylated BSG and mBSG products. Lane 1, BSG; lane 2, mBSG. (B) Low-glycosylated BHc products. Lane 1, BSK. (C) Deglycosylated proBHc. Lane 1, a deglycosylated BHc product derived from deglycosylated BSG; lane 2, a deglycosylated BHc product derived from deglycosylated mBSG; lane 3, a deglycosylated BHc product derived from deglycosylated BSK. M, protein standard.
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f1: Analysis of purified recombinant BHc products by SDS–PAGE.(A) Hyper-glycosylated BSG and mBSG products. Lane 1, BSG; lane 2, mBSG. (B) Low-glycosylated BHc products. Lane 1, BSK. (C) Deglycosylated proBHc. Lane 1, a deglycosylated BHc product derived from deglycosylated BSG; lane 2, a deglycosylated BHc product derived from deglycosylated mBSG; lane 3, a deglycosylated BHc product derived from deglycosylated BSK. M, protein standard.

Mentions: The purified BSG or mBSG expressed in P. pastoris GS115 proteins were visualized by SDS–PAGE as a major band of ~150 kDa and a smear with lesser electrophoretic mobility as a result of hyper-glycosylation (Figure 1A, Table 1). By contrast, the purified BSK protein expressed in P. pastoris GJK0115 appeared by SDS–PAGE as a major band of ~60 kDa because of low-glycosylation (Figure 1B, Table 1).


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

Liu B, Shi D, Chang S, Gong X, Yu Y, Sun Z, Wu J - Sci Rep (2015)

Analysis of purified recombinant BHc products by SDS–PAGE.(A) Hyper-glycosylated BSG and mBSG products. Lane 1, BSG; lane 2, mBSG. (B) Low-glycosylated BHc products. Lane 1, BSK. (C) Deglycosylated proBHc. Lane 1, a deglycosylated BHc product derived from deglycosylated BSG; lane 2, a deglycosylated BHc product derived from deglycosylated mBSG; lane 3, a deglycosylated BHc product derived from deglycosylated BSK. M, protein standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286741&req=5

f1: Analysis of purified recombinant BHc products by SDS–PAGE.(A) Hyper-glycosylated BSG and mBSG products. Lane 1, BSG; lane 2, mBSG. (B) Low-glycosylated BHc products. Lane 1, BSK. (C) Deglycosylated proBHc. Lane 1, a deglycosylated BHc product derived from deglycosylated BSG; lane 2, a deglycosylated BHc product derived from deglycosylated mBSG; lane 3, a deglycosylated BHc product derived from deglycosylated BSK. M, protein standard.
Mentions: The purified BSG or mBSG expressed in P. pastoris GS115 proteins were visualized by SDS–PAGE as a major band of ~150 kDa and a smear with lesser electrophoretic mobility as a result of hyper-glycosylation (Figure 1A, Table 1). By contrast, the purified BSK protein expressed in P. pastoris GJK0115 appeared by SDS–PAGE as a major band of ~60 kDa because of low-glycosylation (Figure 1B, Table 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China 100071.

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.

No MeSH data available.


Related in: MedlinePlus