SECRETION OF FOREIGN PROTEINS FROM SACCHAROMYCES-CEREVISIAE DIRECTED BY ALPHA-FACTOR GENE FUSIONS
SECRETION OF FOREIGN PROTEINS FROM SACCHAROMYCES-CEREVISIAE DIRECTED BY ALPHA-FACTOR GENE FUSIONS
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DOI：
10.1073/pnas.81.17.5330
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
LAI, PH
中科院分区：
文献类型：
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作者：
BITTER, GA;CHEN, KK;LAI, PH
克隆的酵母α-构建了因子结构基因和化学合成的编码人蛋白质类似物的DNA片段。基因融合体编码包括天然α-半胱氨酸的前89个氨基酸的杂合蛋白质。因子前体融合到小的（β-内啡肽，31个氨基酸）或大（α-干扰素，166个氨基酸）外源蛋白。参与α-葡聚糖酶的蛋白水解切割位点在杂合蛋白中，来自天然前体的因子成熟紧接在外源肽之前。α-因子启动子在S.酿酒酵母中，并导致外源蛋白的有效分泌到培养基中。杂交蛋白的加工通过分泌蛋白的氨基酸序列分析来表征。参与α-葡聚糖酶成熟的蛋白水解裂解来自天然前体的因子肽也准确地存在于杂合蛋白中。裂解发生在β-赖氨酸内2个赖氨酸的羧基侧。内啡肽还检测到干扰素蛋白的内部裂解。在这种情况下，裂解以非常低的频率发生，使得> 95%的分泌的干扰素保持完整。
Fusions between the cloned yeast .alpha.-factor structural gene and chemically synthesized DNA segments encoding human protein analogs were constructed. The gene fusions encode hybrid proteins that include the first 89 amino acids of the native .alpha.-factor precursor fused to either a small (.beta.-endorphin, 31 amino acids) or large (.alpha.-interferon, 166 amino acids) foreign protein. Proteolytic cleavage sites involved in .alpha.-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein. The .alpha.-factor promoter was utilized to express the gene fusions in S. cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins was characterized by amino acid sequence analysis of the secreted proteins. The proteolytic cleavages involved in the maturation of .alpha.-factor peptides from the native precursor also occur accurately in the hybrid protein. Cleavages occurred on the carboxyl side of 2 lysines within the .beta.-endorphin peptide. Internal cleavages in the interferon protein were also detected. In this case, the cleavages occurred at a very low frequency such that > 95% of the secreted interferon remained intact.