R&D Group, ASPEX Division, Research Center, Asahi Glass Co., Ltd., 1150 Hazawa-cho, Kanagawa-ku, Yokohama, Japan.
Appl Microbiol Biotechnol. 2010 Jan;85(3):667-77. doi: 10.1007/s00253-009-2151-0. Epub 2009 Aug 11.
Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Delta, vps22Delta, and vps34Delta, were determined on account of their hGH secretion efficiency. The mutant vps10Delta was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.
先前，我们通过删除裂殖酵母 Schizosaccharomyces pombe 中七种阻塞蛋白酶的多个基因，实现了蛋白酶敏感模型蛋白人生长激素 (hGH) 的约 30 倍分泌增强。然而，在产生的多蛋白酶缺陷菌株中发现了分泌 hGH 的细胞内保留。作为一种解决方案，尝试对蛋白酶八重缺失菌株进行与 hGH 细胞内保留相关的细胞内运输途径的遗传修饰。通过将 hGH 与 EGFP 融合进行分泌表达，鉴定出细胞内保留的 hGH 的液泡积累，并且由于其 hGH 分泌效率，确定了三个液泡蛋白分选 (vps)-缺陷菌株 vps10Delta、vps22Delta 和 vps34Delta。突变体 vps10Delta 被发现对 hGH 分泌有效，这表明 vps10 在细胞内保留的 hGH 的液泡积累中起作用。最后，在蛋白酶八重缺失菌株上进行 vps10 删除，导致 hGH 分泌增加约 2 倍。这表明分泌途径修饰和多种蛋白酶缺失可能适用于提高裂殖酵母 S. pombe 中异源蛋白的分泌。
