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Reconstitution of the functional Carotenoid-Binding Protein from silkworm in E. coliстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Slonimskiy Yury B., Egorkin Nikita A., Ashikhmin Aleksandr A., Friedrich Thomas, Maksimov Eugene G., Sluchanko Nikolai N.
- Журнал: International Journal of Biological Macromolecules
- Том: 214
- Год издания: 2022
- Издательство: Elsevier BV
- Местоположение издательства: Netherlands
- Первая страница: 664
- Последняя страница: 671
- DOI: 10.1016/j.ijbiomac.2022.06.135
- Аннотация: Natural water-soluble carotenoproteins are promising antioxidant nanocarriers for biomedical applications. The Carotenoid-Binding Protein from silkworm Bombyx mori (BmCBP) is responsible for depositing carotenoids in cocoons. This determines the silk coloration, which is relevant for sericulture for four thousand years. While BmCBP function is well-characterized by molecular genetics, its structure and carotenoid-binding mechanism remain to be studied. To facilitate this, here we report on successful production of soluble BmCBP in Escherichia coli, its purification and characterization. According to CD spectroscopy and SEC-MALS, this protein folds into a ~ 27-kDa monomer capable of dose-dependent binding of lutein, a natural BmCBP ligand, in vitro. Binding leads to a >10 nm red-shift of the carotenoid absorbance and quenches tryptophan fluorescence of BmCBP. Using zeaxanthin, a close lutein isomer that can be stably produced in engineered E.coli strains, we successfully reconstitute the BmCBP holoform and characterize its properties. While BmCBP successfully matures into the holoform, BmCBP-zeaxanthin complexes are contaminated by the apoform. We demonstrate that the yield of the holoform can be increased by adding bovine serum albumin during cell lysis and that the remaining BmCBP apoform is efficiently removed using hydroxyapatite chromatography. Bacterial production of BmCBP paves the way for its structural studies and applications.
- Добавил в систему: Максимов Евгений Георгиевич