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Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha
S H Shen1, L Bastien, T Nguyen
1Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Abstract:
The methylotrophic yeast, Hansenula polymorpha, has been developed as a host system for the synthesis of heterologous proteins. The middle surface antigen of hepatitis B virus (preS2-HBsAg) has been synthesized under the control of a methanol-regulated promoter derived from the methanol oxidase-encoding gene. The synthesized preS2-HBsAg protein was found to be secreted outside the cell membrane into the periplasm and further excreted into the culture medium following permeabilization of the cell wall with beta-1,3-glucanase (beta Glu). Cell cultures treated with beta Glu were able to continuously synthesize and secrete 22-nm particles of preS2-HBsAg into the medium for several days. The overall yield of antigen from treated cultures was found to be over threefold greater than that of untreated controls. The observation that complex supramolecular structures, such as the 22-nm particles of preS2-HBsAg, can be secreted by H. polymorpha and released into the medium, suggests the potential for these yeasts to be an alternative secretory host.
Insights
Hansenula polymorpha yeast efficiently synthesizes and secretes hepatitis B virus preS2-HBsAg particles. Cell wall permeabilization with beta-1,3-glucanase significantly enhances antigen yield and secretion into the culture medium.
Area of Science:
- Biotechnology
- Microbial Engineering
- Vaccine Development
Background:
- Hansenula polymorpha is a methylotrophic yeast utilized as a host for heterologous protein production.
- The middle surface antigen of hepatitis B virus (preS2-HBsAg) is a target for vaccine development.
Purpose of the Study:
- To develop Hansenula polymorpha as a secretory host for preS2-HBsAg.
- To investigate the effect of cell wall permeabilization on preS2-HBsAg secretion.
Main Methods:
- Utilized a methanol-regulated promoter for preS2-HBsAg synthesis in H. polymorpha.
- Employed beta-1,3-glucanase (beta Glu) to permeabilize the yeast cell wall.
- Quantified preS2-HBsAg particle secretion into the culture medium.
Main Results:
- Synthesized preS2-HBsAg was secreted into the periplasm and subsequently into the medium after beta Glu treatment.
- Beta Glu-treated cultures continuously secreted preS2-HBsAg 22-nm particles for several days.
- Antigen yield was over threefold greater in beta Glu-treated cultures compared to untreated controls.
Conclusions:
- Hansenula polymorpha can be engineered as an effective secretory host for complex protein structures.
- Beta Glu treatment enhances the secretion of preS2-HBsAg particles, indicating potential for large-scale production.
- This yeast system offers a promising alternative for the secretory production of viral antigens.