The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a N-glycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.

中文翻译：

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N-糖基化对巴斯德毕赤酵母中破伤风毒素片段C表达的影响。

巴斯德毕赤酵母蛋白表达系统中发生的N-糖基化过程可能会对酵母分泌的异源糖蛋白的产量产生重大影响。然而，尚未阐明N-糖基化对产率的影响的基础。为了研究N-糖基化对异源蛋白质生产的影响，在破伤风毒素片段C（TetC）的五个潜在的N-糖基化位点上进行了定点突变。在巴斯德毕赤酵母GS115中表达了未改变的TetC（野生TetC）和八个突变体，其中N-糖基化位点的数量和位置发生了改变。重组靶蛋白呈现不同水平的N-糖基化。推测的N-糖基化的野生Tet-C和4个突变位点（4Gly突变体：N280Q）的分泌蛋白水平最高，而假定的N-糖基化位点的1个突变（1Gly突变：N39 / 64/85 / 205Q）具有最高水平的细胞内非分泌异源蛋白。减少天然N-糖基化位点的数量降低了糖基化的水平以及分泌的水平。然而，在位置320处引入N-糖基化位点也降低了重组蛋白的表达和分泌水平。这些结果表明，N-糖基化位点的数目和位置共同影响巴斯德毕赤酵母中异源糖蛋白的表达和分泌。以及分泌水平。然而，在位置320处引入N-糖基化位点也降低了重组蛋白的表达和分泌水平。这些结果表明，N-糖基化位点的数目和位置共同影响巴斯德毕赤酵母中异源糖蛋白的表达和分泌。以及分泌水平。然而，在位置320处引入N-糖基化位点也降低了重组蛋白的表达和分泌水平。这些结果表明，N-糖基化位点的数目和位置共同影响巴斯德毕赤酵母中异源糖蛋白的表达和分泌。