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hCYP2C8 (reductase) |
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Description:
Recombinant human cytochrome P450 2C8 (hCYP2C8) has a broad array of applications ranging from the evaluation of potential pharmaceutical agents to the catalysis of synthetic reactions. Recombinant hCYP2C8 is useful for high throughput screening of drug candidates for possible toxicity, drug-drug interaction and dosage issues. Comparisons of the bacterially expressed human CYP2C8 with the related human liver enzymes give similar kinetic profiles (1). The supplied hCYP2C8 has 11 of the first 17 amino acid residues changed in the N-terminal membrane anchor region that improves expression in E. coli but has not been found to alter enzyme activity (1,2). Human NADPH-cytochrome P450 reductase is also present to permit efficient hCYP2C8 activity.
Source:
The membrane fraction from E. coli DH5a-T1r co-expressing human CYP2C8 and human NADPH-cytochrome P450 reductase.
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
2.8 pmol
Concentration
P450:
2 mM
Total Protein: 1.1 mg/ml
Storage Conditions
Storage Conditions:
100 mM KPO4
0.1 mM EDTA
20% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
Usage notes:- Upon receiving, aliquot and store at -70°C to avoid multiple freeze-thaw cycles. For use thaw in 37°C water bath and immediately place on ice.
- This enzyme was produced using the Pharmazyme™ technology licensed to NEB by PPD Discovery Inc.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
hCYP2C8 Specific Activity::
hCYP2C8 activity was evaluated by following its conversion of paclitaxel to 6α-hydroxypaclitaxel. The reaction buffer was 50 mM potassium phosphate, (pH 7.5) containing 3.3 mM glucose-6-phosphate, 0.4 units/ml glucose-6-phosphate dehydrogenase, 3.3 mM MgCl2, 1.3 mM NADP+ and 20 µM paclitaxel. The addition of hCYP2C8 to a final concentration of 40 nM initiated the assay. At specific time points the reaction was quenched by adding methanol (at 50% of the reaction volume). Formation of 6α-hydroxypaclitaxel was followed by HPLC using a C18 column and a 20 mM ammonium acetate buffer containing 10% methanol and 40% acetonitrile under isocratic conditions. Products and reactants were detected by monitoring their absorbance at 230 nm. The amount of product was determined by comparison to a standard of 6α-hydroxypaclitaxel.
References
- Richardson, T.H. et al. (1995) Arch. Biochem. Biophys., 323, 87-96.
- Iwata, H. et al. (1998) Biochem. Pharamacol., 55, 1315-1325.
- Barnes, H.J. (1996) E. Johnson and M. Waterman (Eds.), Methods Enzymol., 272, pp. 3-14. San Diego: Academic Press.
- Guengerich, F.P. (1991) J. Biol. Chem., 266, 10019-10022.
- Li, A.P. (2001) Drug Discovery Today, 6, 357-366.
Companion Products
E. coli DH5α Control Membranes
hCYP1A2 (reductase)
hCYP2C19 (reductase)
hCYP2D6 (reductase)
hCYP3A4(reductase)
Legal
Licenses/Patents/Disclaimers:
This Product is a Pharmazyme™ isozymes, produced underlicense from PPD Discovery, Inc. (U.S. Patent Nos. 5,240,831 and 5,420,027). Pharmazyme™ is a trademark of PPD, Inc. This product is sold for research use only and not for resale in any form. Commercial use of these products may require a license from PPD Discovery, Inc., Morrisville, NC.
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