Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties

Guo, ZY, Gillam, Emj, Ohmori, S, Tukey, RH and Guengerich, FP (1994) Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties. Archives of Biochemistry and Biophysics, 312 2: 436-446. doi:10.1006/abbi.1994.1330


Author Guo, ZY
Gillam, Emj
Ohmori, S
Tukey, RH
Guengerich, FP
Title Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties
Journal name Archives of Biochemistry and Biophysics   Check publisher's open access policy
ISSN 0003-9861
Publication date 1994-01-01
Year available 1994
Sub-type Article (original research)
DOI 10.1006/abbi.1994.1330
Open Access Status
Volume 312
Issue 2
Start page 436
End page 446
Total pages 11
Place of publication SAN DIEGO
Publisher ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS
Language eng
Subject 1303 Specialist Studies in Education
1304 Biophysics
1312 Molecular Biology
Abstract Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript, alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in similar to 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with K-m values of 0.58 and 15 mu M and V-max values of 8.3 and 2.5 nmol min(-1) (nmol P450 1A1)(-1), respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies. (C) 1994 Academic Press, Inc.
Keyword benzo[a]pyrene
cytochrome P450 1A1
enzyme purification
Escherichia coli
heterologous expression
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID CA44353
ES00267
GM36590
Institutional Status Unknown

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