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AssigneeBoard of Regents, The University of Texas System
InventorsCharles W. Fisher, Henry J. Barnes, Ronald W. Estabrook
Estimating the term…
Disclosed are methods and compositions for effecting bacterial expression of eukaryotic cytochrome P450 enzymes and fusion proteins comprising a eukaryotic P450 domain fused to a reductase enzyme domain in a biologically active form. Certain embodiments involve the expression of eukaryotic cytochrome P450.sub.17.alpha. -hydroxylase which is expressed in large amounts in an E. coli host in a biologically active form without the need for coexpression or admixture of a cytochrome P450 reductase. Methods and compositions are also disclosed for the construction of cytochrome P450 enzyme hybrids or fusion proteins, incorporating the N-terminal 9 amino acids from bovine 17.alpha.-hydroxylase, which will enable many eukaryotic cytochrome P450 enzymes or fusion proteins to be expressed in bacteria.
A DNA segment comprising a gene encoding a fusion protein, said DNA segment being substantially free from total genomic DNA, the fusion protein having a eukaryotic cytochrome P450 hybrid enzyme domain and a cytochrome P450 reductase domain, wherein said gene comprises, in order of transcription, a promoter capable of effecting expression of the encoded fusion protein in a compatible bacterial cell positioned in operable linkage with a DNA region encoding the fusion protein, a ribosome binding site compatible with a bacterial cell, the DNA encoding the fusion protein, and a transcription terminator, wherein the DNA encoding the amino proximal portion of the fusion protein has been modified to allow bacterial expression of the fusion protein in a biologically active form within a bacterial transformant of the Enterobacteriaceae, wherein said eukaryotic cytochrome P450 hybrid enzyme doman has at its amino terminus the nine amino acids Met Ala Leu Leu Leu Ala Val Phe Leu.
A purified fusion protein comprising a eukaryotic cytochrome P450 enzyme domain and a cytochrome P450 hybrid reductase domain, said protein being free of normal eukaryotic glycosylation, wherein said eukaryotic cytochrome P450 hybrid enzyme doman has at its amino terminus the nine amino acids Met Ala Leu Leu Leu Ala Val Phe Leu.
A method for preparing a bacterial cell comprising a DNA segment encoding a fusion protein, the method comprising the steps of:
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