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Isolation, characterization and expression of cDNAs encoding human and marmoset cytochrome P450's

Nanji, Manoj Shantilal; (1995) Isolation, characterization and expression of cDNAs encoding human and marmoset cytochrome P450's. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The expression of individual cytochromes P450 (P450s) has become a valuable tool for studying the structure- function relationship of these proteins and their metabolic capacities. A full-length cDNA encoding human CYP2A6 was expressed both in the baculovirus/Sf9 insect cell system and as a fusion protein with the maltose binding protein (MBP) in Escherichia coli ( E. coli) cells. The expressed proteins were detected by SDS-PAGE and western blotting. MBP-CYP2A6 fusion protein was located in the cell membrane fraction of E. coli cells. In Sf9 insect cells transfected with a recombinant baculovirus, CYP2A6 was located in the microsomal membranes. MBP-CYP2A6 fusion protein expressed in E. coli was functionally inactive towards the CYP2A6 substrate, coumarin. Although the degradation of MBP-CYP2A6 was not detected by western blot analysis, spectral analysis showed a strong P420 component suggesting misfolding of the polypeptide due to the interaction with the MBP domain. Attempts to purify MBP-CYP2A6 from E. coli were not successful. Baculovirus expressed CYP2A6 was found to be enzymatically active towards the metabolism of coumarin but not testosterone. Endogenous NADPH-cytochrome P450 reductase in Sf9 cells did interact with the expressed CYP2A6. However, the amounts of this protein were not sufficient for the amount of CYP2A6 expressed and catalytic studies required the addition of exogenous NADPH-cytochrome P450 reductase to obtain maximum CYP2A6 activity. CYP2A6 was purified from Sf9 cells by affinity chromatography. A cDNA encoding a CYP2A was isolated from marmoset liver total RNA by reverse transcription and PCR. When compared to the sequence of CYP2A6, marmoset CYP2A cDNA contained a deletion of a nucleotide C after the initiation codon which changed the reading frame. Although not useful for heterologous studies, the cDNA was used as a probe for northern blot analysis of marmoset liver total RNA isolated from the livers of untreated or phenobarbital treated animals. CYP2A mRNA was induced 20-fold on treatment of marmoset with phenobarbital.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Isolation, characterization and expression of cDNAs encoding human and marmoset cytochrome P450's
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10102849
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