Abstract
Using the Trc99A/P450scc vector, a plasmid was constructed in which cDNAs for cytochrome P450scc, adrenodoxin reductase, and adrenodoxin are situated in a single expression cassette. This plasmid was shown to direct the synthesis of all the above proteins in Escherichia coli. Their localization in the E. coli cells and stoichiometry were determined. Cell homogenates exhibited cholesterol hydroxylase/lyase activity, due to catalytically active forms of all three proteins. Thus, the full set of constituents of the mammalian cholesterol hydroxylase/lyase system was shown to be synthesized in bacterial cells for the first time.
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Abbreviations
- Ad:
-
adrenodoxin
- AdR:
-
adrenodoxin reductase
- CH/L:
-
cholesterol hydroxylase/lyase
- DTT:
-
1,4-dithiothreitol
- IPTG:
-
isopropyl-β-D-thiogalactoside
- P450scc:
-
cytochrome P450scc
- PMSF:
-
phenylmethylsulfonyl fluoride
- RBS:
-
ribosome-binding site
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Original Russian Text © T. V. Shashkova, V. N. Luzikov, L. A. Novikova, 2006, published in Biokhimiya, 2006, Vol. 71, No. 7, pp. 996–1001.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM06-030, June 11, 2006.
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Shashkova, T.V., Luzikov, V.N. & Novikova, L.A. Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells. Biochemistry (Moscow) 71, 810–814 (2006). https://doi.org/10.1134/S0006297906070145
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DOI: https://doi.org/10.1134/S0006297906070145