Tominaga Akihiro, Kotani Tetsuya, Watanabe Masahiro, Takegawa Kaoru
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Yokohama Technical Center, AGC Inc., Kanagawa 230-0045, Japan.
J Biosci Bioeng. 2025 Oct;140(4):237-243. doi: 10.1016/j.jbiosc.2025.07.003. Epub 2025 Jul 31.
The fission yeast Schizosaccharomyces pombe has been often used as a host for heterologous protein production; however, a method for extracellular secretion of heterologous protein would be advantageous for ease of purification and for native protein structure. In a previous study, overexpression of endogenous protein disulfide isomerase (PDI) genes improved the secretion of recombinant human transferrin in S. pombe. In the present study, we have explored whether Pdi1 can be used for the secretion of heterologous proteins in S. pombe. Overexpression of a fusion protein of Pdi1p and the heterologous protein EGFP (Pdi1p-EGFP), in the host S. pombe A8 strain, which lacks eight intracellular and extracellular proteases, resulted in efficient extracellular secretion of the fusion protein. To identify the optimal region of Pdi1p for use as an extracellular carrier, we compared the secretion of EGFP fused to the N-terminal Pdi1p signal domain and deletion mutants of Pdi1p. The signal sequence alone did not improve secretion, but deletion of two domains at the C-terminus did improve secretion. Notably, the x domain was important for secretion of the fusion protein. As a result of these findings, we have established a system for efficient secretion of target heterologous proteins by using optimally designed Pdi1p as a carrier for extracellular secretion.
裂殖酵母粟酒裂殖酵母常被用作异源蛋白生产的宿主；然而，一种异源蛋白细胞外分泌的方法对于简化纯化过程和保持天然蛋白结构将是有利的。在先前的一项研究中，内源性蛋白二硫键异构酶（PDI）基因的过表达提高了粟酒裂殖酵母中重组人转铁蛋白的分泌。在本研究中，我们探究了Pdi1是否可用于粟酒裂殖酵母中异源蛋白的分泌。在缺乏八种细胞内和细胞外蛋白酶的宿主粟酒裂殖酵母A8菌株中，过表达Pdi1p与异源蛋白EGFP的融合蛋白（Pdi1p-EGFP），导致该融合蛋白有效地细胞外分泌。为了确定用作细胞外载体的Pdi1p的最佳区域，我们比较了与Pdi1p N端信号域融合的EGFP以及Pdi1p缺失突变体的分泌情况。单独的信号序列并不能改善分泌，但在C端缺失两个结构域确实改善了分泌。值得注意的是，x结构域对于融合蛋白的分泌很重要。基于这些发现，我们建立了一个系统，通过使用经过优化设计的Pdi1p作为细胞外分泌载体来高效分泌目标异源蛋白。
