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

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Дата последнего поиска статьи во внешних источниках: 19 декабря 2017 г.

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[1] Expression of cholesterol hydroxylase/lyase system proteins in yeast s. cerevisiae cells as a self-processing polyprotein / V. S. Efimova, L. V. Isaeva, D. S. Makeeva et al. // Molecular Biotechnology. — 2017. — Vol. 59, no. 9-10. — P. 394–406. 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. Keywords Cytochrome P450 CYP11A1 Adrenodoxin Adrenodoxin reductase FMDV 2A peptide Heterologous expression. [ DOI ]

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