Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.
The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.
D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D005814:Genes, Viral; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D006023:Glycoproteins; D014759:Viral Envelope Proteins