Polycistronic expression of mitochondrial steroidogenic P450scc system in the nonsteroidogenic HEK293T cell line

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.