Abstract：
Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.
摘要：
嗜热四膜虫是重组蛋白生产的替代生物。然而,嗜热梭菌的生产效率很低，主要是由于富含半胱氨酸蛋白酶。在这项研究中,我们研究了在嗜热菌培养物中补充E-64抑制剂是否可以提高重组蛋白的生产效率而没有任何毒副作用。我们的研究表明，在30°C和38°C的烧瓶培养中，补充E-64对嗜热T细胞没有致死作用。使用分泌组作为E-64补充细胞培养物的蛋白酶来源进行的体外蛋白酶活性分析显示，使用牛血清白蛋白减少了蛋白质底物的降解，利妥昔单抗,和乳乳球蛋白。E-64还在一定水平上阻止了重组产生和分泌的TtmCherry2-sfGFP融合蛋白的蛋白水解。E-64的这种降低的抑制作用可能是由于抑制的蛋白酶的遗传补偿。因此,发现5µM浓度的E-64是一种无毒的蛋白酶抑制性补充剂，可提高嗜热T.这项研究表明，使用E-64可以通过在培养过程中连续降低细胞外半胱氨酸蛋白酶活性来提高细胞外重组蛋白生产的效率。
