Sato T, Uemura H, Izumoto Y, Nakao J, Nakamura Y, Matsubara K
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Gene. 1989 Nov 30;83(2):355-65. doi: 10.1016/0378-1119(89)90122-4.
The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.
酵母酿酒酵母在酵母PHO5启动子的控制下表达编码人唾液α-淀粉酶（Amy）的克隆cDNA，并将活性酶分泌到培养基中。采用了两种方法来确定Amy中对于正确分泌和糖基化所必需的部分。在一种方法中，构建了嵌合体，将多种分泌信号序列（酵母交配因子前体序列、酵母酸性磷酸酶信号序列和人胃泌素信号序列）与缺失分泌信号的Amy cDNA融合。另一种方法涉及分析Amy编码区的一系列缺失序列和一系列点突变。结果表明，异源信号序列足以在酵母中实现正确分泌，而与一些额外氨基酸的插入无关。在大多数情况下，具有缺失和Cys-465取代的酶即使具有完整的分泌信号序列也不会被分泌。相反，它们以糖基化形式在细胞中积累。因此，正确的分泌似乎需要待分泌的多肽部分具有合适的构象。
