Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.
Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.
Belloc
C
C
Institut National de la Santé et de la Recherche Médicale Unité 75, Université René Descartes, Paris, France.
Baird
S
S
Cosme
J
J
Lecoeur
S
S
Gautier
J C
JC
Challine
D
D
de Waziers
I
I
Flinois
J P
JP
Beaune
P H
PH
D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D000918:Antibody Specificity; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000032:analysis; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating