Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae.
The cloning and expression of the hepatitis B middle-protein surface antigen gene in 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. Immunoblotting 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.
Langley
K E
KE
Amgen Inc., Thousand Oaks, CA 91320.
Egan
K M
KM
Barendt
J M
JM
Parker
C G
CG
Bitter
G A
GA
D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D013194:Staining and Labeling; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D000939:Epitopes; D006514:Hepatitis B Surface Antigens