Methods and compositions for the expression of biologically active eukaryotic cytochrome P45OS in bacteria
AssigneeBoard of Regents, The University of Texas
InventorHenry J. Barnes
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Abstract
Disclosed are methods and compositions for effecting bacterial expression of eukaryotic cytochrome P450 enzymes in a biologically active form. Particular embodiments involve the expression of eukaryotic cythochrome 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. Techniques are disclosed for effecting improved bacterial expression which are proposed to be useful in connection with all eukaryotic cytochrome P450 enzymes which one may desire to express in bacterial hosts such as E. coli and others.
Claims49
A stably transformed bacterial cell of the family Enterobacteriaceae which expresses a biologically active cytochrome P450 enzyme, said transformant comprising a DNA segment consisting essentially of a cytochrome P450 expression construct maintaining host-compatible transcriptional and translational regulatory DNA sequence elements in operable linkage with a eukaryotic cytochrome P450 coding region, said expression construct comprising a transcription promoter, ribosome binding site, spacer region and transcription terminator.
The bacterial cell of claim or 5, wherein the codon encoding the second amino acid of the cytochrome P450 enzyme comprises GCT, AAA, ACC, ACT, TAT, AAT, AAC, CAC, CAT, CGT, AGA, CGC, TTT, ATC, ATA, ATT, GTA, TTG, CTT, CTC, TTA, GCC, GCA, GAA, AGC, or AGT.
A method for the production of a biologically active cytochrome P450 enzyme in a recombinant bacterium comprising preparing a bacterial cell in accordance with anyone of claims 1 through 23 and culturing said cell under condition appropriate to effect expression of the cytochrome P450 enzyme.
An isolated DNA segment consisting essentially of a cytochrome P450 expression construct maintaining host-compatible transcriptional and translational regulatory DNA sequence elements in operable linkage with a eukaryotic cytochrome P450 coding region, said expression construct comprising a transcription promoter, ribosome binding site, spacer region and transcription terminator.
The DNA segment of claim 30, wherein the cytochrome P450 enzyme is a member of the cytochrome P450 I, II, III, IV, VI, XIA, XIB, XVII, XIX, XXI or XXVI family.
The DNA segment of claim 32, wherein the cytochrome P450 enzyme is a 17.alpha.-hydroxylase.
A method for obtaining bacterial expression of a biologically active eukaryotic cytochrome P450 enzyme comprising the steps of:
- (a) obtaining a DNA segment which comprises a selected eukaryotic cytochrome P450 enzyme gene;
- (b) modifying the segment so as to operatively combine the gene with a bacterially compatible ribosome binding site, spacer region, transcription terminator and promoter to form a bacterial expression unit;
- (c) transforming bacteria with said bacterial expression unit forming transformant colonies;
- and
- (d) selecting a transformant colony which expresses said gene in a biologically active manner;
- and
- (e) culturing said selected transformant under conditions effective to express said eukaryotic cytochrome P450 enzyme.
The method of claim 49, wherein said segment is modified by combining said gene with a bacterial expression cassette.
Dependencies are inferred from the claim text.