Expression of Cytochrome P450 2D6 in Escherichia coli, Purification, and Spectral and Catalytic Characterization

Gillam, Emj, Guo, ZY, Martin, MV, Jenkins, CM and Guengerich, FP (1995) Expression of Cytochrome P450 2D6 in Escherichia coli, Purification, and Spectral and Catalytic Characterization. Archives of Biochemistry and Biophysics, 319 2: 540-550. doi:10.1006/abbi.1995.1329


Author Gillam, Emj
Guo, ZY
Martin, MV
Jenkins, CM
Guengerich, FP
Title Expression of Cytochrome P450 2D6 in Escherichia coli, Purification, and Spectral and Catalytic Characterization
Journal name Archives of Biochemistry and Biophysics   Check publisher's open access policy
ISSN 0003-9861
Publication date 1995-06-10
Year available 1995
Sub-type Article (original research)
DOI 10.1006/abbi.1995.1329
Open Access Status
Volume 319
Issue 2
Start page 540
End page 550
Total pages 11
Publisher ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS
Language eng
Subject 1312 Molecular Biology
1304 Biophysics
1303 Specialist Studies in Education
Abstract Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated. We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW. These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed. Production of holoprotein was highly dependent upon the addition of δ-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system. The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either. A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield, Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1′-hydroxylation). The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses.
Keyword Biochemistry & Molecular Biology
Biophysics
Biochemistry & Molecular Biology
Biophysics
BIOCHEMISTRY & MOLECULAR BIOLOGY
BIOPHYSICS
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID CA44353
ES07028
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: ResearcherID Downloads - Archived
 
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