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Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3

Item Type:Article
Title:Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3
Creators Name:Scheller, U. and Kraft, R. and Schroeder, K.L. and Schunck, W.H.
Abstract:The cytosolic domain of microsomal P450 52A3 (P450Cm1) was isolated as a soluble and functionally active protein. The NH2-terminal region that anchors the P450 protein to the endoplasmic reticulum was removed by sequence-specific proteolysis at a designed cleavage site. For that purpose, P450Cm1 was genetically engineered to establish at position 63-66 the sequence Ile-Glu-Gly-Arg, which is recognized by the restriction protease factor Xa. The modified P450 was produced in high yields as an integral membrane protein in Saccharomyces cerevisiae. In the microsomal fraction, it was accessible to factor Xa digestion, releasing a readily soluble, shortened P450 protein. For large scale preparation of the cytosolic domain, the modified P450Cm1 was first purified and then subjected to sequence-specific proteolysis. The highly purified delta(1-66)P450Cm1 exhibited unchanged spectral characteristics and catalyzed the hydroxylation of n-hexadecane with 85% of the activity determined for full-length wild-type P450Cm1. The method developed for the preparation of the cytosolic domain of P450Cm1 may be more generally applicable to facilitate structure-function studies on membrane-bound P450 forms.
Keywords:Amino Acid Sequence, Base Sequence, Catalysis, Cell Membrane, Cytochrome P-450 Enzyme System, DNA, Hydrolysis, Liquid Chromatography, Microsomes, Molecular Cloning, Molecular Sequence Data, Peptide Fragments, Polyacrylamide Gel Electrophoresis, Recombinant Proteins, Saccharomyces Cerevisiae, Spectrum Analysis, Substrate Specificity
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:269
Number:17
Page Range:12779-12783
Date:29 April 1994
Official Publication:http://www.jbc.org/content/269/17/12779.abstract
PubMed:View item in PubMed

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