Schultz L D, Tanner J, Hofmann K J, Emini E A, Condra J H, Jones R E, Kieff E, Ellis R W
Gene. 1987;54(1):113-23. doi: 10.1016/0378-1119(87)90353-2.
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.
爱泼斯坦-巴尔病毒的主要包膜糖蛋白（gp350）已在酿酒酵母中表达并分泌为一种400 kDa的糖蛋白。这是酵母中分泌如此大的、高度糖基化的异源蛋白的首个例子。由于gp350对酿酒酵母具有高毒性，最初的细胞生长需要抑制gp350的表达。利用温度或半乳糖诱导型启动子，细胞能够生长，然后诱导gp350表达。诱导后，糖蛋白在细胞内以及培养基中积累。只有糖基化程度最高的形式被分泌，这表明N-连接聚糖在指导分泌中起作用。酵母来源蛋白的O-连接糖基化程度与成熟病毒gp350的相似。N-连接糖基化根据培养条件和所用宿主菌株略有不同，且比与成熟病毒gp350相关的更广泛。尽管没有证据表明重组质粒表达的糖蛋白的mRNA不止一种，但诱导后早期酵母中积累了各种大小的糖蛋白，这可能反映了糖基化的中间体。酵母来源的糖蛋白与针对gp350的动物和人多克隆抗体以及针对gp350的中和鼠单克隆抗体发生反应，这表明该糖蛋白保留了天然糖蛋白的几个表位。
