Idiris Alimjan, Tohda Hideki, Bi Ke-wei, Isoai Atsushi, Kumagai Hiromichi, Giga-Hama Yuko
ASPEX Division, Research Center, Asahi Glass Co., Ltd., Yokohama 221-8755, Japan.
Appl Microbiol Biotechnol. 2006 Nov;73(2):404-20. doi: 10.1007/s00253-006-0489-0. Epub 2006 Jun 27.
The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83-99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.
构建蛋白酶缺陷型突变体以避免产物降解是目前用于提高异源蛋白表达的生产力和分泌效率的策略之一。我们之前通过分别破坏52个蛋白酶基因构建了一组粟酒裂殖酵母的单蛋白酶缺陷型突变体，并且成功鉴定出了有用的破坏株（伊迪里斯等人，《酵母》23:83 - 99，2006年）。在本研究中，我们尝试对13个蛋白酶基因进行多重缺失，其中单个基因的缺失先前已被证实有利于减少细胞外产物的降解。利用基于聚合酶链反应的基因替换技术，通过最多破坏7个蛋白酶基因构建了一系列多重缺失菌株。然后通过对一种蛋白水解敏感的模型蛋白——人生长激素（hGH）的实际表达来测定所得多重缺失菌株对异源表达的影响。每个所得突变体分泌hGH的时间曲线表明，多重蛋白酶缺失处理显著提高了hGH的生产力。数据清楚地表明，在裂殖酵母中破坏多个蛋白酶基因是控制特别易受蛋白酶作用的异源蛋白的蛋白水解降解的有效方法。
