Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene |
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Authors: | Linda Owers Narhi Long-Ping Wen Armand J. Fulco |
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Affiliation: | (1) Department of Biological Chemistry, UCLA School of Medicine, and Laboratory of Biomedical and Environmental Sciences, University of California, 90024 Los Angeles, California, USA;(2) Present address: AMGen, Thousand Oaks, CA, USA;(3) Laboratory of Biomedical and Environmental Sciences, University of California, 900 Veteran Avenue, 90024-1786 Los Angeles, California, USA |
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Abstract: | Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this polypeptide (cytochrome P-450BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450BM-3 from B. megaterium and putative P-450BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS Sodium Dodecylsulfate - PAGE Polyacrylamide Gel Electrophoresis - HPLC High Performance Liquid Chromatography |
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Keywords: | Cytochrome P-450BM-3 Bacillus megaterium Escherichia coli protein charachterization recombinant DNA monoxygenase |
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