Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae

  • Langley K
  • Egan K
  • Barendt J
 et al. 
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Abstract

The cloning and expression of the hepatitis B middle-protein surface antigen gene m the yeast Saccharomyces cerevisiae is described. A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used. Expressed material, in the form of supramolecular particles, was purified and characterized. Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host. This proteolysis was substantially reduced by utilization of a protease-deficient host. Immuno-blotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present. All species were analyzed by N-terminal sequencing after electroelution from gels. Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms. Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types. Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues. The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region. © 1988.

Author-supplied keywords

  • Vaccine
  • glycosylation
  • proteolysis
  • recombinant DNA

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Authors

  • Keith E. Langley

  • Kevin M. Egan

  • Jean M. Barendt

  • Carol G. Parker

  • Grant A. Bitter

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