Abstract
In this review we describe the use of targeted antibodies developed to facilitate studies on the expression of P450 proteins. The challenge of producing specific antibodies that distinguish between often highly related P450 proteins has led to the development of methods of antibody production to meet this need. Targeting antibodies toward the C terminus of P450 proteins has been found to be a particularly successful approach that is both rapid and efficient at producing specifically binding antibodies. Recent advances in genomic sequencing and proteomics now allow ready identification of expressed proteins. The levels and distributions of these proteins may be determined using antibody-based methods. However, for each protein to be studied, a unique antibody will be required. Consequently, some means of producing large numbers of well defined antibodies is needed. In this context, the potential of extending the approach used to produce specific antibodies against P450 proteins to the wider field of functional genomics is discussed.
Footnotes
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↵2 We have also produced a CYP3A7-specific antibody by targeting an internal sequence of CYP3A7. This antibody has been used to examine the expression of CYP3A7 in the liver of 59 normal human donors by immunoblotting. In 58 of these, no evidence of expression was found. In one sample an immunoreactive protein was present, but we have yet to confirm whether this is CYP3A7 (unpublished data). Thus, although CYP3A7 mRNA is expressed at detectable levels in the majority of adult livers (Schuetz et al. 1994; Burk et al. 2002), this does not appear to be the case for CYP3A7 protein.
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Donald Davies received a Bachelor's degree in chemistry in 1962 from the University of Wales (UK). In 1965 he received a Ph.D. degree in biochemistry from St. Mary's Hospital Medical School, University of London under the supervision of Professor Tecwyn Williams for studies of drug metabolism.
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From 1965 to 1967 Professor Davies was a Visiting Fellow in the Laboratory of Chemical Pharmacology at the National Institutes of Health (United States) where he was a member of Dr. Gillette's Section working on mechanisms of drug oxidation in the very early days of research on P450. In 1967 he returned to the UK to join the Department of Clinical Pharmacology at the Royal Postgraduate Medical School (London, UK) and was appointed Professor of Biochemical Pharmacology in 1980. In 1987 he succeeded Professor Colin Dollery as head of the department (at what is now Imperial College London). Recently, he resigned from this post to become Director of Research at ML Laboratories plc but continues to be a member of the professorial staff at Imperial College London. A major element in his research on mechanisms of drug action and toxicity has been his studies of human P450 enzymes, an interest he developed while working with Dr. Gillette.
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Alan Boobis received a Bachelor's degree in pharmacology in 1971 from the University of Glasgow (UK). In 1974 he received a Ph.D. degree in pharmacology from the University of Glasgow (UK) under the supervision of Garth Powis working in the area of drug metabolism. His doctoral work involved studies of the influence of changes in hemodynamics in the liver on drug disposition and the effects of lipid peroxidation on P450 integrity. In 2003, he received an OBE for his work on the risk assessment of pesticides.
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Professor Boobis then worked as a Fogarty Visiting Fellow for two years with Dr. Dan Nebert at the National Institutes of Health (Bethesda, MD). During this time he studied the effects of modulating factors on benzo-[a[pyrene DNA adduction and was also involved in an investigation of genetic and developmental factors on P450 expression and activity. In 1976 he joined the department of Clinical Pharmacology at what was then the Royal Postgraduate Medical School (London, UK), as a Medical Research Council research training fellow under the supervision of Professor Donald Davies. He was then appointed to a tenured lectureship and subsequently promoted to Professor of Biochemical Pharmacology, his current position (at what is now Imperial College London). Recently, he also took over as director of the Department of Health Toxicology Unit at Imperial College London. His current research interests include the regulation of human P450 enzymes by genetic and environmental factors and the development of novel biomarkers of toxicity.
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Robert Edwards received a Bachelor's degree in applied biochemistry in 1977 from Brunel University (UK). In 1982 he received a Ph.D. degree in biochemistry from Guy's Hospital Medical School, University of London. In 1984 he joined the Department of Clinical Pharmacology at the Royal Postgraduate Medical School (London, UK) to work with Alan Boobis and Donald Davies on the characterization of P450 enzymes. Here, he developed an anti-peptide approach for the production of antibodies against the major forms of hepatic P450 expressed in human and other species. In 2000 he was appointed as Research Lecturer at Imperial College London. His current research activities include the application of the anti-peptide antibody approach to proteomics as well as a continuing interest in the characterization of P450 enzymes by immunochemical methods.
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↵1 Abbreviation used is: P450, cytochrome P450.
- Received April 15, 2003.
- Accepted June 25, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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A STRATEGY FOR INVESTIGATING THE CYP SUPERFAMILY USING TARGETED ANTIBODIES IS A PARADIGM FOR FUNCTIONAL GENOMIC STUDIES
