Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line
Efimova V. S., Isaeva L. V., Labudina A. A., Tashlitsky V. N., Rubtsov M. A., Novikova L. A.
Journal of Cellular Biochemistry
Vol.120, Issue3, P. 3124-3136
Опубликовано: 2019
Тип ресурса: Статья
Аннотация:
The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substr
Ключевые слова:
adrenodoxin; adrenodoxin reductase; CYP11A1; cytochrome P450; foot-and-mouth disease virus 2A peptide; HEK293; heterologous expression; steroidogenesis
3(or 17)beta hydroxysteroid dehydrogenase; cholesterol monooxygenase (side chain cleaving); complementary DNA; fusion protein; pregnenolone; 3(or 17)beta hydroxysteroid dehydrogenase; adrenodoxin; cholesterol monooxygenase (side chain cleaving); ferredoxin nicotinamide adenine dinucleotide phosphate reductase; pregnenolone; Article; carboxy terminal sequence; cytosol; enzyme activity; flow cytometry; fluorescence microscopy; genetic transfection; HEK293T cell line; high performance liquid chromatography; human; human cell; human cell culture; mitochondrion; plasmid; polycistronic expression; priority journal; protein expression; protein localization; steroidogenesis; genetics; HEK293 cell line; metabolism; mitochondrion; Western blotting; 3-Hydroxysteroid Dehydrogenases; Adrenodoxin; Blotting, Western; Cholesterol Side-Chain Cleavage Enzyme; Chromatography, High Pressure Liquid; Ferredoxin-NADP Reductase; Flow Cytometry; HEK293 Cells; Humans; Microscopy, Fluorescence; Mitochondria; Pla
Язык текста: Английский
ISSN: 1097-4644
Efimova V. S.
Isaeva L. V.
Labudina A. A.
Tashlitsky V. N.
Rubtsov M. A. Mikhail Aleksandrovich 1983-
Novikova L. A.
Ефимова В. С.
Исаева Л. В.
Лабудина А. А.
Ташлицкy В. Н.
Рубцов М. А. Михаил Александрович 1983-
Новикова Л. А.
Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line
Текст визуальный непосредственный
Journal of Cellular Biochemistry
John Wiley & Sons
Vol.120, Issue3 P. 3124-3136
2019
Статья
adrenodoxin adrenodoxin reductase CYP11A1 cytochrome P450 foot-and-mouth disease virus 2A peptide HEK293 heterologous expression steroidogenesis
3(or 17)beta hydroxysteroid dehydrogenase cholesterol monooxygenase (side chain cleaving) complementary DNA fusion protein pregnenolone 3(or 17)beta hydroxysteroid dehydrogenase adrenodoxin cholesterol monooxygenase (side chain cleaving) ferredoxin nicotinamide adenine dinucleotide phosphate reductase pregnenolone Article carboxy terminal sequence cytosol enzyme activity flow cytometry fluorescence microscopy genetic transfection HEK293T cell line high performance liquid chromatography human human cell human cell culture mitochondrion plasmid polycistronic expression priority journal protein expression protein localization steroidogenesis genetics HEK293 cell line metabolism mitochondrion Western blotting 3-Hydroxysteroid Dehydrogenases Adrenodoxin Blotting, Western Cholesterol Side-Chain Cleavage Enzyme Chromatography, High Pressure Liquid Ferredoxin-NADP Reductase Flow Cytometry HEK293 Cells Humans Microscopy, Fluorescence Mitochondria Pla
The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substr