Bitter G A, Chen K K, Banks A R, Lai P H
Proc Natl Acad Sci U S A. 1984 Sep;81(17):5330-4. doi: 10.1073/pnas.81.17.5330.
Fusions between the cloned yeast alpha-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been 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 Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins has been 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. In addition, cleavages occurred on the carboxyl side of two lysines within the beta-endorphin peptide. Internal cleavages in the interferon protein were also detected. However, in this case, the cleavages occurred at a very low frequency such that greater than 95% of the secreted interferon remained intact.
已构建了克隆的酵母α-因子结构基因与编码人蛋白质类似物的化学合成DNA片段之间的融合体。这些基因融合体编码杂合蛋白，其中包括天然α-因子前体的前89个氨基酸与小的（β-内啡肽，31个氨基酸）或大的（α-干扰素，166个氨基酸）外源蛋白融合。在杂合蛋白中，参与从天然前体成熟为α-因子的蛋白水解切割位点紧邻外源肽之前。利用α-因子启动子在酿酒酵母中表达基因融合体，结果使外源蛋白有效地分泌到培养基中。已通过对分泌蛋白的氨基酸序列分析来表征杂合蛋白的加工过程。从天然前体成熟为α-因子肽所涉及的蛋白水解切割在杂合蛋白中也能准确发生。此外，在β-内啡肽肽段内两个赖氨酸的羧基侧发生了切割。还检测到干扰素蛋白的内部切割。然而，在这种情况下，切割发生的频率非常低，以至于超过95%的分泌型干扰素保持完整。
