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hCYP2D6 (reductase) |
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Description:
Recombinant human cytochrome P450 2D6 (hCYP2D6) has a broad array of applications ranging from the evaluation of potential pharmaceutical agents to the catalysis of synthetic reactions. Recombinant hCYP2D6 is useful for high throughput screening of drug candidates for possible toxicity, drug-drug interaction and dosage issues. Bacterially expressed human CYP2D6 and CYP2D6 purified from human liver have similar kinetic profiles (1,2). The supplied hCYP2D6 has 5 of the first 8 amino acid residues changed in the N-terminal membrane anchor region to improve expression in E. coli but has not been found to alter enzyme activity (2). Human NADPH-cytochrome P450 reductase is also present to permit efficient hCYP2D6 activity.
Source:
The membrane fraction from E. coli DH5a-T1r co-expressing human CYP2D6 and human NADPH-cytochrome P450 reductase.
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
14 AFU/min
Concentration
P450:
1 μM
Total Protein: 5 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.
hCYP2D6 Assay Conditions::
hCYP2D6 activity was evaluated by following its conversion of 3-cyano-7-ethoxycoumarin (CEC) to 3-cyano-7-hydroxycoumarin (CHC). The reaction buffer consisted of 50 mM potassium phosphate (pH 7.5) containing 3.3 mM glucose-6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, 5 mM MgCl2, 1 mM NADPH and 50 µM CEC. At time zero hCYP2D6 (60 nM final concentration) was added to reaction buffer prewarmed to 37°C. The reaction was monitored at 37°C using a Perkin-Elmer LS50B fluorimeter using an excitation wavelength of 420 nm (7.5 nm slit width) and an emission wavelength of 490 nm (7.5 nm slit width). The reaction rate was calculated from the linear portion of the progress curve.
References
- Gillam, E.M.J. et al. (1995) Arch. Biochem. Biophys., 319, 540-550.
- Barnes, H.J., unpublished observations.
- 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)
hCYP2C8 (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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