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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Continuous assay to monitor reactivation of various oxygenases from E. coli inclusion bodiesтезисы доклада
- Авторы: Zakharyants A.A., Hushpulian D.M., Smirnova N.A., Poloznikov A.A., Moroz N.A., Tishkov V.I., Gazaryan I.G.
- Сборник: Program and Abstract Book of International Conference "Biocatalysis-2015: Fundamentals and Applications", Moscow region, Avantel Club Istra
- Тезисы
- Год издания: 2015
- Место издания: Innovations and High Technologies MSU Ltd Moscow
- Первая страница: 106
- Последняя страница: 106
- Аннотация: HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) belong to alpha-ketoglutarate de-pendent non-heme iron dioxygenases catalyzing proline hydroxylation in HIF. PHD2 has been reacti-vated by refolding from inclusion bodies. To optimize the refolding procedure a novel continuous spec-trophotometric assay has been developed by monitoring ferrocyanide oxidation accelerated in the pres-ence of HIF protein or peptide substrate. Comparison of the soluble enzyme with the one reactivated from inclusion bodies demonstrates a 4-5-fold higher specific activity of the latter (5 mol/min/vol) as-sayed by formation of a fluorescent adduct of ketoglutarate with o-phenylene diamine. FAD-monoxygenase domain of murine MICAL1 was expressed in E. coli and reactivated from inclusion bod-ies using a ferricyanide reduction assay. Despite both enzyme are unstable in the absence of DTT, the novel assays developed allow one to easily optimize refolding and purification protocols. This work was supported by Russian Foundation for Basic Research, grant 13-04-01909-а.
- Добавил в систему: Тишков Владимир Иванович