Tokunaga M, Wada N, Hishinuma F
Mitsubishi-Kasel Institute of Life Sciences, Tokyo, Japan.
Nucleic Acids Res. 1988 Aug 11;16(15):7499-511. doi: 10.1093/nar/16.15.7499.
Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.
携带线性双链DNA质粒pGKL1和pGKL2的酿酒酵母分泌一种由97千道尔顿、31千道尔顿和28千道尔顿亚基组成的杀伤毒素（《核酸研究》，15卷，1031 - 1046页，1987年）。我们分离出了编码28K前体蛋白N端前导序列的DNA，并构建了一种新的酿酒酵母分泌载体。与28K信号序列融合的小鼠α -淀粉酶被高效分泌到培养基中，其效率与融合到交配因子α和97K - 31K杀伤信号序列的情况相似。这些数据清楚地表明28K前导序列起到了分泌信号的作用。在培养基中检测到了糖基化和非糖基化的α -淀粉酶分子。α -淀粉酶的分泌被sec18 - 1突变阻断。从培养基中回收的分泌型α -淀粉酶在SDS -聚丙烯酰胺凝胶中的迁移速度比体外合成的α -淀粉酶前体形式更快。这些证据表明，与28K杀伤信号序列融合的小鼠α -淀粉酶是通过酵母的正常分泌途径进行加工、糖基化和分泌的。
