Wilgoss, Amanda Louise;
(2005)
Expression and Characterisation of Modified Human
Cytochrome P450 IB l (CYPIBI).
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Cytochromes P450 (CYPs, P450s) are a superfamily of haem-containing enzymes, responsible for the metabolism of a diverse collection of both endogenous compounds and xenobiotics. An increased level of CYPIBI protein in tumour tissues makes this particular isoform a potential target in cancer therapeutics. In order to guide development of new compounds, CYPIBI must be fully characterised and the structure of the active site defined. CYPIBI is expressed naturally at low levels, thus a method to generate recombinantly expressed protein as a more abundant source was developed. The CYPIBI gene was amplified from a cDNA clone, using PCR. The PCR primers were designed to modify the CYPIBI gene sequence so that expression was facilitated in E. coli. Two engineered forms of CYPIBI were cloned since CYPs are integral membrane proteins and purification and solubilisation is difficult. One generated a modified, membrane bound protein (CYP1B1"MEM) and the other generated a membrane anchor- free protein (CYPIBIoSOL). CYPlBl"SOL was developed in order to facilitate purification as a soluble protein. Removal of the membrane anchor region resulted in a membrane associated protein, which was removed from the membranes by washing with 0.01% Triton X-100, and was further isolated using metal chelate chromatography. Activity analysis was carried out using the 7-ethoxyresorufin O-deethylation assay, initially with resuspended bacterial membranes contining either the membrane associated CYP1B1 *SOL or intrinsic CYP1B1 *MEM. Despite the membrane anchor region being removed, the activity of CYPlBl*SOL was not affected when compared with the activity of CYP1B1*MEM. The activity of CYP1B1"S0L was comparable to both CYPIBI oMEM and the commercially available native CYPIBI. The N-terminal membrane-spanning region is therefore, not vital for the activity of CYPIBI since membrane-associated CYPIBI oSOL retained 100% of the membrane bound activity.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Expression and Characterisation of Modified Human Cytochrome P450 IB l (CYPIBI) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | Health and environmental sciences; CYP1B1; Cytochrome |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10116674 |
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