title: Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha.
abstract: An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
authors.affiliation: RheinBiotech GmbH, Düsseldorf, F.R.G.
authors.forename: Z A
authors.initials: ZA
authors.lastname: Janowicz
authors.forename: K
authors.initials: K
authors.lastname: Melber
authors.forename: A
authors.initials: A
authors.lastname: Merckelbach
authors.forename: E
authors.initials: E
authors.lastname: Jacobs
authors.forename: N
authors.initials: N
authors.lastname: Harford
authors.forename: M
authors.initials: M
authors.lastname: Comberbach
authors.forename: C P
authors.initials: CP
authors.lastname: Hollenberg
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003470:Culture Media; D004271:DNA, Fungal / Q000737:chemistry; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015183:Restriction Mapping
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011994:Recombinant Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen