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L A Novikova - One of the best experts on this subject based on the ideXlab platform.
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polycistronic expression of the mitochondrial steroidogenic p450scc system in the HEK293T Cell Line
2019Co-Authors: Vera S Efimova, L V Isaeva, Anastasia Andreevna Labudina, Vadim N Tashlitsky, Mikhail A Rubtsov, L A NovikovaAbstract: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 substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T Cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T Cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of Cells expressing all three CHL system components, and to that of nature steroidogenic Cells. Thus, the P450scc activity detected in Cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T Cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T Cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
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polycistronic expression of mitochondrial steroidogenic p450scc system in the nonsteroidogenic HEK293T Cell Line
2016Co-Authors: Anastasia Andreevna Labudina, Vera S Efimova, L V Isaeva, Mikhail A Rubtsov, L A NovikovaAbstract:Сholesterol hydroxylase/lyase system (CH/L) consists of cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR) and initiates mammalian steroidogenesis converting cholesterol to pregnenolone. FMDV 2A-Based method allows to express multiple proteins from single transcript. Ribosome skips one peptide bond in C-terminus of the 2A conservative sequence, synthesizing individual protein products. Polycistronic vector pcDNA3.1_СH/L_2A_GFP created includes cDNAs, located in the transcriptional unit in order: preCoxIVP450scc-2A-Adx-2A-AdR-GFP. Vector contains cDNA for P450scc with CoxIV mitochondrial targeting presequence. Confocal microscopy of HEK-293T Cells, transfected with created vector pcDNA3.1_СH/L_2A_GF and control Cells transfected with plasmid comprising only GFP cDNA, shows the change of GFP fluorescent signal localization from nuclear to mitochondrial and correlation between signals from GFP and MitoTracker in HEK_CH/L. Western-blotting also shows the presence of CH/L proteins in homogenate and mitochondria of HEK_CH/L Cells. Expressed heterologous proteins form functionally active system. Activity of P450scc in vivo is 83 ng of pregnenolone/ml of medium for 24 h in the presence of 20α-hydroxycholesterol (HPLC data), and in vitro is 32.7 ng/mg of mitochondrial protein for 1 h (ELISA). Therefore, the active P450-system in mammalian Cells was firstly reconstructed using 2A peptid technology that can be used for fundamental research.
Vera S Efimova - One of the best experts on this subject based on the ideXlab platform.
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polycistronic expression of the mitochondrial steroidogenic p450scc system in the HEK293T Cell Line
2019Co-Authors: Vera S Efimova, L V Isaeva, Anastasia Andreevna Labudina, Vadim N Tashlitsky, Mikhail A Rubtsov, L A NovikovaAbstract: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 substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T Cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T Cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of Cells expressing all three CHL system components, and to that of nature steroidogenic Cells. Thus, the P450scc activity detected in Cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T Cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T Cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
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polycistronic expression of mitochondrial steroidogenic p450scc system in the nonsteroidogenic HEK293T Cell Line
2016Co-Authors: Anastasia Andreevna Labudina, Vera S Efimova, L V Isaeva, Mikhail A Rubtsov, L A NovikovaAbstract:Сholesterol hydroxylase/lyase system (CH/L) consists of cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR) and initiates mammalian steroidogenesis converting cholesterol to pregnenolone. FMDV 2A-Based method allows to express multiple proteins from single transcript. Ribosome skips one peptide bond in C-terminus of the 2A conservative sequence, synthesizing individual protein products. Polycistronic vector pcDNA3.1_СH/L_2A_GFP created includes cDNAs, located in the transcriptional unit in order: preCoxIVP450scc-2A-Adx-2A-AdR-GFP. Vector contains cDNA for P450scc with CoxIV mitochondrial targeting presequence. Confocal microscopy of HEK-293T Cells, transfected with created vector pcDNA3.1_СH/L_2A_GF and control Cells transfected with plasmid comprising only GFP cDNA, shows the change of GFP fluorescent signal localization from nuclear to mitochondrial and correlation between signals from GFP and MitoTracker in HEK_CH/L. Western-blotting also shows the presence of CH/L proteins in homogenate and mitochondria of HEK_CH/L Cells. Expressed heterologous proteins form functionally active system. Activity of P450scc in vivo is 83 ng of pregnenolone/ml of medium for 24 h in the presence of 20α-hydroxycholesterol (HPLC data), and in vitro is 32.7 ng/mg of mitochondrial protein for 1 h (ELISA). Therefore, the active P450-system in mammalian Cells was firstly reconstructed using 2A peptid technology that can be used for fundamental research.
Anastasia Andreevna Labudina - One of the best experts on this subject based on the ideXlab platform.
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polycistronic expression of the mitochondrial steroidogenic p450scc system in the HEK293T Cell Line
2019Co-Authors: Vera S Efimova, L V Isaeva, Anastasia Andreevna Labudina, Vadim N Tashlitsky, Mikhail A Rubtsov, L A NovikovaAbstract: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 substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T Cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T Cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of Cells expressing all three CHL system components, and to that of nature steroidogenic Cells. Thus, the P450scc activity detected in Cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T Cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T Cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
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polycistronic expression of mitochondrial steroidogenic p450scc system in the nonsteroidogenic HEK293T Cell Line
2016Co-Authors: Anastasia Andreevna Labudina, Vera S Efimova, L V Isaeva, Mikhail A Rubtsov, L A NovikovaAbstract:Сholesterol hydroxylase/lyase system (CH/L) consists of cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR) and initiates mammalian steroidogenesis converting cholesterol to pregnenolone. FMDV 2A-Based method allows to express multiple proteins from single transcript. Ribosome skips one peptide bond in C-terminus of the 2A conservative sequence, synthesizing individual protein products. Polycistronic vector pcDNA3.1_СH/L_2A_GFP created includes cDNAs, located in the transcriptional unit in order: preCoxIVP450scc-2A-Adx-2A-AdR-GFP. Vector contains cDNA for P450scc with CoxIV mitochondrial targeting presequence. Confocal microscopy of HEK-293T Cells, transfected with created vector pcDNA3.1_СH/L_2A_GF and control Cells transfected with plasmid comprising only GFP cDNA, shows the change of GFP fluorescent signal localization from nuclear to mitochondrial and correlation between signals from GFP and MitoTracker in HEK_CH/L. Western-blotting also shows the presence of CH/L proteins in homogenate and mitochondria of HEK_CH/L Cells. Expressed heterologous proteins form functionally active system. Activity of P450scc in vivo is 83 ng of pregnenolone/ml of medium for 24 h in the presence of 20α-hydroxycholesterol (HPLC data), and in vitro is 32.7 ng/mg of mitochondrial protein for 1 h (ELISA). Therefore, the active P450-system in mammalian Cells was firstly reconstructed using 2A peptid technology that can be used for fundamental research.
Mikhail A Rubtsov - One of the best experts on this subject based on the ideXlab platform.
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polycistronic expression of the mitochondrial steroidogenic p450scc system in the HEK293T Cell Line
2019Co-Authors: Vera S Efimova, L V Isaeva, Anastasia Andreevna Labudina, Vadim N Tashlitsky, Mikhail A Rubtsov, L A NovikovaAbstract: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 substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T Cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T Cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of Cells expressing all three CHL system components, and to that of nature steroidogenic Cells. Thus, the P450scc activity detected in Cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T Cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T Cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
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polycistronic expression of mitochondrial steroidogenic p450scc system in the nonsteroidogenic HEK293T Cell Line
2016Co-Authors: Anastasia Andreevna Labudina, Vera S Efimova, L V Isaeva, Mikhail A Rubtsov, L A NovikovaAbstract:Сholesterol hydroxylase/lyase system (CH/L) consists of cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR) and initiates mammalian steroidogenesis converting cholesterol to pregnenolone. FMDV 2A-Based method allows to express multiple proteins from single transcript. Ribosome skips one peptide bond in C-terminus of the 2A conservative sequence, synthesizing individual protein products. Polycistronic vector pcDNA3.1_СH/L_2A_GFP created includes cDNAs, located in the transcriptional unit in order: preCoxIVP450scc-2A-Adx-2A-AdR-GFP. Vector contains cDNA for P450scc with CoxIV mitochondrial targeting presequence. Confocal microscopy of HEK-293T Cells, transfected with created vector pcDNA3.1_СH/L_2A_GF and control Cells transfected with plasmid comprising only GFP cDNA, shows the change of GFP fluorescent signal localization from nuclear to mitochondrial and correlation between signals from GFP and MitoTracker in HEK_CH/L. Western-blotting also shows the presence of CH/L proteins in homogenate and mitochondria of HEK_CH/L Cells. Expressed heterologous proteins form functionally active system. Activity of P450scc in vivo is 83 ng of pregnenolone/ml of medium for 24 h in the presence of 20α-hydroxycholesterol (HPLC data), and in vitro is 32.7 ng/mg of mitochondrial protein for 1 h (ELISA). Therefore, the active P450-system in mammalian Cells was firstly reconstructed using 2A peptid technology that can be used for fundamental research.
L V Isaeva - One of the best experts on this subject based on the ideXlab platform.
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polycistronic expression of the mitochondrial steroidogenic p450scc system in the HEK293T Cell Line
2019Co-Authors: Vera S Efimova, L V Isaeva, Anastasia Andreevna Labudina, Vadim N Tashlitsky, Mikhail A Rubtsov, L A NovikovaAbstract: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 substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T Cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T Cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of Cells expressing all three CHL system components, and to that of nature steroidogenic Cells. Thus, the P450scc activity detected in Cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T Cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T Cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
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polycistronic expression of mitochondrial steroidogenic p450scc system in the nonsteroidogenic HEK293T Cell Line
2016Co-Authors: Anastasia Andreevna Labudina, Vera S Efimova, L V Isaeva, Mikhail A Rubtsov, L A NovikovaAbstract:Сholesterol hydroxylase/lyase system (CH/L) consists of cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR) and initiates mammalian steroidogenesis converting cholesterol to pregnenolone. FMDV 2A-Based method allows to express multiple proteins from single transcript. Ribosome skips one peptide bond in C-terminus of the 2A conservative sequence, synthesizing individual protein products. Polycistronic vector pcDNA3.1_СH/L_2A_GFP created includes cDNAs, located in the transcriptional unit in order: preCoxIVP450scc-2A-Adx-2A-AdR-GFP. Vector contains cDNA for P450scc with CoxIV mitochondrial targeting presequence. Confocal microscopy of HEK-293T Cells, transfected with created vector pcDNA3.1_СH/L_2A_GF and control Cells transfected with plasmid comprising only GFP cDNA, shows the change of GFP fluorescent signal localization from nuclear to mitochondrial and correlation between signals from GFP and MitoTracker in HEK_CH/L. Western-blotting also shows the presence of CH/L proteins in homogenate and mitochondria of HEK_CH/L Cells. Expressed heterologous proteins form functionally active system. Activity of P450scc in vivo is 83 ng of pregnenolone/ml of medium for 24 h in the presence of 20α-hydroxycholesterol (HPLC data), and in vitro is 32.7 ng/mg of mitochondrial protein for 1 h (ELISA). Therefore, the active P450-system in mammalian Cells was firstly reconstructed using 2A peptid technology that can be used for fundamental research.