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[Heterologous expression of eukaryotic CYP450. 1. Heterologous expression of cytochrome P450 2B4 using groups with various affinity in E. coli]статья
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: Zhgun A.A., El’darov M.A., Solodar’ L.I., Sokolov N.N., Archakov A.I., Skriabin K.G.
- Журнал: Voprosy medit∎sinsko∎ khimii
- Том: 47
- Номер: 4
- Год издания: 2001
- Издательство: Rossi∎skai∎a akademii∎a medit∎sinskikh nauk
- Местоположение издательства: Moskva, Russia (Federation)
- Первая страница: 382
- Последняя страница: 392
- Аннотация: The expression levels of cytochrome P450 2B4 variants with N- and C-terminal modifications were compared and some of the enzymatic characteristics of recombinant proteins studied. Following C-terminal hybrids for CYP2B4 gene were constructed: 1) with intein-chitin binding domain cassette 2) with hexahistidine tag. These modifications were combined with P450 2B4 glutathione-S-transferase N-terminal fusions [Pernecky S.J., et. al., (1995) Arch. Biochem. Biophys., 318, 446-456]. The obtained constructs provided for the synthesis of full-length protein products in E. coli cells with holoenzyme yield at the levels of 200-1000 nmoles/l of the bacterial culture. Partial in vivo proteolysis was observed for C-terminal fusions with intein moiety despite the presence of glycine aminoacid residue at the junction of two proteins. The principle inapplicability of standard purification scheme for isolation of P450 2B4-intein fusions is demonstrated, since the P450 domain is inactivated at 40 mM DTT concentrations. The recombinant full-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control of T7 promoter and purified using immobilized metal-ion chelating chromatography. The C-terminal hexahistidine tag does not affect the catalytic properties of recombinant enzyme in 7-pentoxyresorufin O-dealkylation reaction.
- Добавил в систему: Эльдаров Михаил Анатольевич