Yeliz Dündar Orhan , Ayça Fulya Üstüntanır Dede , Şeyma Duran , Muhittin Arslanyolu
{"title":"利用 E-64 半胱氨酸蛋白酶抑制剂在嗜热四膜虫中生产重组蛋白","authors":"Yeliz Dündar Orhan , Ayça Fulya Üstüntanır Dede , Şeyma Duran , Muhittin Arslanyolu","doi":"10.1016/j.ejop.2024.126085","DOIUrl":null,"url":null,"abstract":"<div><p><em>Tetrahymena thermophila</em> is an alternative organism for recombinant protein production. However, the production efficiency in <em>T. thermophila</em> is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to <em>T. thermophila</em> 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 <em>T. thermophila</em> cells in flask culture at 30 °C and 38 °C. <em>In vitro</em> 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 <em>T. thermophila</em>. 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<em>.</em></p></div>","PeriodicalId":12042,"journal":{"name":"European journal of protistology","volume":"94 ","pages":"Article 126085"},"PeriodicalIF":1.9000,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila\",\"authors\":\"Yeliz Dündar Orhan , Ayça Fulya Üstüntanır Dede , Şeyma Duran , Muhittin Arslanyolu\",\"doi\":\"10.1016/j.ejop.2024.126085\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p><em>Tetrahymena thermophila</em> is an alternative organism for recombinant protein production. However, the production efficiency in <em>T. thermophila</em> is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to <em>T. thermophila</em> 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 <em>T. thermophila</em> cells in flask culture at 30 °C and 38 °C. <em>In vitro</em> 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 <em>T. thermophila</em>. 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<em>.</em></p></div>\",\"PeriodicalId\":12042,\"journal\":{\"name\":\"European journal of protistology\",\"volume\":\"94 \",\"pages\":\"Article 126085\"},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2024-04-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"European journal of protistology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S093247392400035X\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"European journal of protistology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S093247392400035X","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila
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.
期刊介绍:
Articles deal with protists, unicellular organisms encountered free-living in various habitats or as parasites or used in basic research or applications. The European Journal of Protistology covers topics such as the structure and systematics of protists, their development, ecology, molecular biology and physiology. Beside publishing original articles the journal offers a forum for announcing scientific meetings. Reviews of recently published books are included as well. With its diversity of topics, the European Journal of Protistology is an essential source of information for every active protistologist and for biologists of various fields.