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Cytochrome P450scc system performs the first rate-limiting stage of steroidogenesis in mammals. The bovine P450scc system was reconstructed in Saccharomyces cerevisiae, using a foot-and-mouth disease virus 2A peptide (F2A)-based construct, to co-express cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). During the translation of the self-processing fusion protein P450scc-F2A-Adx-F2A-AdR, the first and the second linkers are cleaved with different efficiencies (96 % and 11 %, respectively), resulting in the unbalanced expression of individual proteins. The low cleavage efficiency and the relative Adx and AdR protein levels were increased through replacing the second F2A peptide with different sequences and changing the order of Adx and AdR. The P450scc, AdR, and Adx sequences located upstream of the F2A affected F2A processing, to various degrees. Moreover, using molecular dynamics (MD) simulations, we showed that the 2A peptide fused to the C-terminus of Adx formed the steric hindrance during enzymatic complex formation, resulting in the reduction of catalytic activity. Thus, the functional activity of the reconstructed P450scc system was determined not only by the efficiency of 2A peptides but also by the overall sequence of the expressed 2A-polyprotein. Our results can be applied to the development of 2A-based co-translation strategies, to produce other multicomponent protein systems. |
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