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Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein

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Abstract

2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins—cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

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Acknowledgements

This study was supported by RFBR, Grant Number: 15-08-00721. We acknowledge A.I. Luneva for her assistance in analyzing protein expression in yeast cells.

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Authors and Affiliations

  1. Department of Molecular Biology, Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, Russia, 119234

    Vera S. Efimova & Mikhail A. Rubtsov

  2. LIA 1066 French-Russian Joint Cancer Research Laboratory, Villejuif, France

    Vera S. Efimova & Mikhail A. Rubtsov

  3. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

    Ludmila V. Isaeva & Ludmila A. Novikova

  4. Faculty of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University, Moscow, Russia

    Desislava S. Makeeva

  5. Department of Biochemistry, I.M. Sechenov First Moscow State Medical University, Moscow, Russia

    Mikhail A. Rubtsov

  6. Strategic Management Department, I.M. Sechenov First Moscow State Medical University, Moscow, Russia

    Mikhail A. Rubtsov

  7. LIA 1066 French-Russian Joint Cancer Research Laboratory, Moscow, Russia

    Vera S. Efimova & Mikhail A. Rubtsov

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  1. Vera S. Efimova
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  2. Ludmila V. Isaeva
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Correspondence to Vera S. Efimova.

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Efimova, V.S., Isaeva, L.V., Makeeva, D.S. et al. Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein. Mol Biotechnol 59, 394–406 (2017). https://doi.org/10.1007/s12033-017-0028-5

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