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