Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha.
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.
Janowicz
Z A
ZA
RheinBiotech GmbH, Düsseldorf, F.R.G.
Melber
K
K
Merckelbach
A
A
Jacobs
E
E
Harford
N
N
Comberbach
M
M
Hollenberg
C P
CP
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
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