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Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolutionстатья
Информация о цитировании статьи получена из
Web of Science
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 29 мая 2015 г.
- Авторы: Koksharov M.I., Ugarova N.N.
- Журнал: Luminescence : the journal of biological and chemical luminescence
- Том: 25
- Номер: 2
- Год издания: 2010
- Издательство: John Wiley & Sons Inc.
- Местоположение издательства: United States
- Первая страница: 135
- Последняя страница: 136
- DOI: 10.1002/bio.1211
- Аннотация: Firefly luciferase catalyzes the bioluminescent reaction of firefly luciferin oxidation in the presence of ATP, Mg2+ and molecular oxygen. Due to the high quantum yield of bioluminescence, catalytic activity and substrate specificity this enzyme is widely used in methods based on ATP determination, as a luminescent biomarker in molecular biology, in pyrosequencing and as a label for immunoassays. However, rapid inactivation of the wild-type (WT) luciferases at the temperatures above 30°C often limits their applications. Luciferase from Luciola mingrelica fireflies displays low thermostability loosing about 50% of activity within 50 minutes at 37°C. In this work we used consecutive cycles of random PCR mutagenesis to increase luciferase thermostability. The mutant S118C was used as a starting point due to its higher thermostability than that of WT (1). Mutagenesis was applied to a part of the gene coding 130-391 out of 548 amino acid residues of L. mingrelica luciferase. The ease of in vivo luciferase bioluminescence detection and the ability of E. coli cells to withstand elevated temperatures up to 55°C allowed us to identify thermostable mutants by a simple non-lethal in vivo screening of a large number of E. coli colonies containing mutant luciferases. The scheme used allowed to carry out each cycle of mutagenesis in a rapid and efficient manner. Four consecutive rounds of random PCR mutagenesis and screening led to significant improvement of luciferase thermostability without compromising its activity (2). The resulting mutant was designated 4TS. It contains contains 8 point mutations and has a half-life at 42°C of 10 h, a 66-fold increase over WT (Fig. 1). Moreover, specific activity of 4TS is 1.9-fold higher compared to WT. The analysis of substitutions shows that 4 mutations are mainly responsible for the increased thermostability: R211L, A217V, E356L and S364C. Mutations of R211 and S364 residues of firefly luciferase are reported for the first time. Firefly luciferase is usually used at temperatures up to 37°C. 4TS retains 70% activity after two days of incubation at 37°C (Fig. 1) so its stability is sufficient for most common applications.
- Добавил в систему: Кокшаров Михаил Иванович
Прикрепленные файлы
| № | Имя | Описание | Имя файла | Размер | Добавлен |
|---|---|---|---|---|---|
| 1. | Полный текст | Abstract_Koksharov.pdf | 129,2 КБ | 11 ноября 2012 [koksharov83] | |
| 2. | Полный текст | Презентация на конференции (pdf) | ISBC-2010_Koksharov_presentation.pdf | 1,2 МБ | 11 ноября 2012 [koksharov83] |