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CYTOCHROME P450
RECOMBINANT ENZYMES
(see also our special line of native enzymes)
see also below the recombinant human NADPH-P450 Oxidoreductase & Cytochrome b5
Cytochrome P450, Recombinant Human,
Purified & Purified Reconstituted Form ,or Microsomes
-10% discount on 5 or more any combination (except **)
BULK QUOTES ON REQUEST
P450 Isoform Product#
Format
Quantity Price
IA2 RDI-P2294 Purified 150 ug $375.00
IA2 RDI-P2304 Reconstituted (see below) $562.00
3A4 RDI-P2194 Purified 150 ug $375.00
3A4 RDI-P2305 Reconstituted (see below) $1000.00 **
3A4
RDI-P2377
Microsomes 0.5nmol
$242.00
new
cat#: 30R-AC031
3A5
RDI-P2512
microsomes 0.5nmol
$242.00
new
cat#: 30R-AC033
2C9 RDI-P2355 Purified 100ug $375.00
2C9
RDI-P2378
Microsomes 0.5nmol
$242.00
new
cat#: 30R-AC030
2C9 RDI-P2362 Reconstituted (see below) $625.00
2C19 RDI-P2570 Microsomes 0.5nmol $219.00
2D6 RDI-P2283 Microsomes 0.5nmol $219.00
2D6 RDI-P2774 purified (*) 150ug $375.00
2E1
RDI-P2223
Purified 150 ug
$412.50
new
cat#: 30R-AC037
2E1 RDI-P2306 Reconstituted (see below) $1000.00 **
P450 control
RDI-P2315 negative microsome control 5mg $242.00
**
new
cat#: 30R-AT014
Applications
* In vitro Drug Metabolism & toxicology Research
* Stereo-and/or Regio-specific Hydroxylation Reactions
Description
The hepatic microsomal cytochrome P450s are members of a superfamily of monoocygenases
that catalyze the Phase I metabolism of xenobiotics, the initial step in the
biotransformation and elimination of a wide variety of drugs and environmental
pollutants.These P450s are located primarily in the liver and are integrated
into the membrane of the endophasmic reticulum.The difficulty and tediousness
of isolating and reconstituting the native enzymes from
human tissue has hampered their use for drug metabolism studies with well-defined
in vitro sytems. Recent development of methods for high level expression of
active, mammalian P450s with baculovirus and E. coli has enabled RDI to offer
recombinant human P450 isoforms in three different formats: Soluble purified
enzymes, purified enzymes in a reconstituted format, and recombinant
microsomes.
Soluble, purified, recombinant human P450s are produced in E.coli. All have
been modified to some degree at the N-termainal to allow expression in E.coli,
but these changes have not caused any discernible changes in substrate
specificity or catalytic properties. The purified enzymes are available either
as separatecomponents or in a reconstituted format with oxidoreductase,
cytochrome b5, and the appropriate lipids, which offers the advantages of using
highly purified enzymes. These microsomes are produced from insect cells
coinfected with a baculovirus vector expressing both a P450 isoform and rabbit
NADPH-P450 oxidoreductase. This format is very simple to use and requires no
additional protein components for full enzymatic activity. The turnover numbers
for P450s in both formates are similar to that seen using human liver
microsomes.
Purified P450s in
Reconstituted Form
RDI provides some purified P450s in a ready-to-use reconstituted form that
includes a complete enzyme mix and separate reaction buffer. This "Mix and
Metabolize" configuration greatly simplifies the use of purified
cytochrome P450 isozymes with their associated protein, making them as easy to
use as microsomes but with the inherent advantage of purified proteins. The
amount of the three contained in the reconstituted format varies for each P450
isoform version. For example, the 3A4 reconstituted version contains a mixture
of 3750 pmol, P450 protein, 7500 pmol NADPH-P450 oxidoreductase,and 3750 pmol
cytochrome b5. In all cases, however, each version contains sufficient protein
to perform 50-100 asays. The amount of enzyme mix and the volume used for an
assay will depend on several factors which include:
The sensitivity of the analytical assay for the metabolites and products from
catalysis, the kinetic parameter of enzyme catalysis for a particular substrate
and P450 if known, the incubation period of the assay, the solubility of the
test compound or substrate, or the specific preferences of individual research
groups.
*Please note that some purified P450's are available as either an
individual purified isoform or with the isoform included in the easy-to-use
Reconstituted Form. The reconstituted version contains two solutions: Solution
1: Enzyme Mix, which contains a mixture of recombinant P450 protein, NADPH-P450
oxidoreductase, and cytochrome b5 in an optimized buffer solution,and Soluation
2- The Reaction Buffer. A "Mix and Metabolize" protocol is also
included with each kit. The amount of the three proteins contained in the
reconstituted format varies for each P450 isoform version. For example, the
P450 3A4 reconstituted version contains 3750 pmol P450 protein, 7500 pmol
oxidoreductase, and 3750 pmol cytochrome b5. In all cases, however, each
version contains sufficient protein to perform 50-100 assays, depending on the
specific application.
CLICK HERE to View a
sample protocol and references for using CYP's in reconstituted form
Click here to view a sample
protocol and references for using CYP's in microsome form.
SAMPLE SPECIFICATIONS: (see batch sheet with each shipment for batch
specific information)
Cytochrome P450 Specifications:
Isoform 1A2
Format Purified *
Concentration: 0.8 mg/ml in 120 mM potassium phosphate pH 7.4, 0.1 mM EDTA, 0.05 mM DTT, 20% (v/v) glycerol
Guaranteed Amount of CYP450: > 1500 pmol
Specific Content: > 10 nmol spectral P450 per milligram of protein.
Source: Human recombinant protein produced from overexpressing plasmid in E.col. N-terminal modifications were as pre viously described (Sandhu et al. 1994, Arch.Biochem. Biophys. 309 168-177).
Specific Activity: Ethoxyresorufin: > 250 pmol/min/nmol ,
Methoxyresorufin: > 500 pmol/min/nmol
Isoform 3A4
Format: Purified*
Concentration: >0.8 mg/ml in 20mM potassium phosphate pH 7.4,0.2
mM EDTA, 1mM DTT, 20% (v/v) glycerol
Guaranteed Amount of CYP450: > 1500 pmol
Specific Content: > 10 nmol spectral P450 per milligram of protein.
Source: Human recombinant protein produced from overexpressing plasmid in E.coli. N-terminal modifications were as previously described (Gillam et al. 1993,Arch.Biochem.Biophys. 305 123-131)
Specific Activity: Erythromycin: > 3 nmol/min/nmol, Testosterone:
> 12 nmol/min/nmol
Isoform: 3A4
Format: Microsomes
Concentration: > 20mg/ml in 100mM potassium phosphate pH 7.5,0.1 mM EDTA, 1mM DTT, 20% (v/v) glycerol.
Guaranteed Amount of CYP450: > 750 pmol
Specific Content: > 30 pmol spectral P450 per milligram of protein.
Source: Microsomes prepared from insect cells that were infected with a baculovirus containing the cDNA for human CYP3A4 and rabbit cytochrome P450 oxidoreductase (CYPOR).
Specific Activity: Testosterone: > 4.5 nmol/min/mg.
Isoform: 2C19
Format: Microsomes
Concentration: > 20 mg/ml in 100mM potassium phosphate pH 7.5.0.1 mM EDTA, 1mM DTT, 20% (v/v) glycerol.
Guaranteed Amount of CYP450: >2000 pmol
Specific Content: > 80 pmol spectral P450 per milligram of protein.
Source: Microsomes prepared from insect cells that were infected with a baculovirus containing the cDNA for human CYP3A4 and rabbit cytochrome P450 oxidoreductase (CYPOR)
Specific Activity: Mephenytoin: > 400 pmo/min/mg
Isoform: 2D6
Format: Microsomes
Concentration: >20 mg/ml in 100 mM potassium phosphate pH 7.5,0.1 mM EDTA, 1mM DTT, 20% (v/v) glycerol.
Guaranteed Amount of CYP450: >250 pmol
Specific Content: > 10 pmol spectral P450 per milligram of protein.
Source: Microsomes prepared from insect cells that were infected with a baculovirus containing the cDNA for human CYP2D6 (valine at position 374) and rabbit cytochrome P450 oxidoreductase (CYPOR).
Specific Activity: Bufuralol: > 1 nmol/min/mg.
Isoform: 2E1
Format: Purified*
Concentration: >0.8 mg/ml in 50 mM Tris HC1 pH 7.7,0.1 mM EDTA, 0.05 mM DTT. 20% (v/v) glycerol.
Guaranteed Amount of CYP450: > 1500 pmol.
Specific Content: > 10 nmol spectral P450 per milligram of protein.
Source: Human recombinant protein produced from overexpressing plasmid in E.coli. N-terminal modifications were as previously described (Gillam et.al. 1994, Arch.Biochem.Biophys. 312 59-66).
Specific Activity: N-Nitrosodimethylamine: > 8 nmol/min/nmol, Chlorzoxazone: > 5 nmol/min/nmol.
All isoforms should be stored at -80'C.
Additional DRUG METABOLIZING ENZYMES (for use with the above CYP450's)
DRUG METABOLIZING ENZYMES (REVISED) NEW HUMAN
Recombinant MATERIAL
Product # Source Quantity Price
P2309 HUMAN 200 ug $375.00
-Bulk Quotes on request
NADPH-P450 Oxidoreductase Purified,Recombinant HUMAN
-replaces older discontinued rat material-
Applications
* For use with in vitro P450-dependent Biotransformation Reactions.
* In Vitro Drug Metabolism & Toxicology Research
Description
NADPH-P450 Oxidoreducatase (OR) transfers electrons form NADPH to the various isoforms of cytochrome P450; it is typically used at a 1:1 or 2:1 ratio to P450 for in vitro reconstitution assays. It is a membrane-bound flavoprotein, containing one mol each of FAD and FMN. The new RDI product is HUMAN Oxidoreductase produced from a recombinant baculovirus. . Also see our native human oxidoreductase.
Sample Specifications Source: Recombinant (insect cells infected with a
baculovirus)
Concentration: 1.2mg/ml in 10mM potassium phosphate, pH 7.7, 0.1 mM EDTA, 1mM
DTT ,20% glycerol.
Physical Purity: Greater than 90% as assessed by visualization of 5 ug
of protein on Coomassic-stained SDS gel
Activity: 24 umol cytochrome c reduced per minute per milligram protein at room
temperature. Using a MW of
76.5 kDa, 6.4ul=100 pmoles.
Storage: -80 DEG C long term. Avoid repeated freeze thaw cycles.
References
1. Yamano,S.,Aoyama,T.,McBride,O.W.,Hardwick,J.P.,Geiboin,H.V., Gonzalez,F.J.,(1989) Molec.Pharmacol 35: 83-88
2. Schenkman,J.,Greim,H.,eds.(1993) Handbook of Experimental Pharmacology, vol. 105.
3. Shen,A.L., Porter,T.D.,
CYTOCHROME b5
Product# Source
Quantity Price
RDI-P2252 Human
100 ug $412.50
new cat#: 30R-AC036
Applications
* For use with in vitro P450-dependent Biotransformation Reactions
* In Vitro Drug Metabolism & Toxicology Research.
Description
Cytochrome b5 is a membrane-bound hemoprotein that enhances the catalytic
efficiency of some P450 isoforms,most notably members of the 3A subfamily/ It
is not clear whether it plays a direct role in electron transfer or an indirect
role through allosteric interactions with the P450. RDI Cytochrome b5 is
overexpressed from the human cDNA in E. coli, purified to homogeneity, and
supplied in a soluble format. Quality control includes testing theability of
the enzyme to stimulate testosterone 6B-hydroxylation in a reconstituted
system with human P450 3A4. See also our native expressed cytochrome
b5.
Specifications
Source: Recombinant E. coli.
Concentration: 0.5-10 mg/ml
Physical Purity: Greater than 90% as assessed by visualization of 10 mg of protein on a Coomassie-stained SDS
Quantitation: Heme content measured using difference spectrophotomctry. An extinction coefficient of gel 118 mM-1 cm-1 for the absorbance difference between 490 and 424 nm is used to calculate the concentration of Cytochrom b5
References:
1. Gillam,F.M.J., Baba,T.,Kim,B.R.,Ohmon,S., Guengerich,F.P.,(1993) Arch. Biochem.Biophys. 305: 123-131
2. Holomans,P.J. Shet.M.S.
Precaution: For in vitro research use only-not for use in or on humans or animals-not
for use in diagnostics. Not responsible for any patent infringements with the
use or derivation of these products.
See also our various
antibodies for use with many of the native and recombinant CYP450's
RDI Division of Fitzgerald Industries Intl
phone (800) 370-2222 or
(978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com
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