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Thermostabilization of firefly luciferase by in vivo directed evolutionстатья
Информация о цитировании статьи получена из
Web of Science,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.
- Авторы: Koksharov M.I., Ugarova N.N.
- Журнал: Protein Engineering, Design and Selection
- Том: 24
- Номер: 11
- Год издания: 2011
- Издательство: Oxford University Press
- Местоположение издательства: United Kingdom
- Первая страница: 835
- Последняя страница: 844
- DOI: 10.1093/protein/gzr044
- Аннотация: Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42 degrees C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.
- Добавил в систему: Кокшаров Михаил Иванович
Прикрепленные файлы
| № | Имя | Описание | Имя файла | Размер | Добавлен |
|---|---|---|---|---|---|
| 1. | Supplementary materials | Supplementary_material.pdf | 1,4 МБ | 11 ноября 2012 [koksharov83] | |
| 2. | Полный текст | Free link to full text (pdf, html) | Koksharov_Ugarova_PEDS_2011_link-vs.pdf | 51,2 КБ | 14 ноября 2012 [koksharov83] |