Recombinant Human DHFR Protein, CF

R&D Systems | Catalog # 8456-DR

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human DHFR Protein (8456-DR)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P00374

Applications

Enzyme Activity
View all Dihydrofolate Reductase/DHFR Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Dihydrofolate Reductase/DHFR protein
Met1-Asp187, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met1 & Val2

Predicted Molecular Mass

22 kDa

SDS-PAGE

23 kDa, reducing conditions

Activity

Measured by the reduction of dihydrofolic acid (DHF).
The specific activity is >5,500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8456-DR
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Dihydrofolate Reductase/DHFR

Dihydrofolate Reductase (DHFR) is an approximately 21 kDa enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, which is crucial for the synthesis of purines, thymidylate, and certain amino acids (1-4). Structurally, DHFR consists of an eight-stranded beta -sheet and four alpha -helices (2). Human DHFR shares 90% amino acid identity with mouse and rat DHFR. In addition to acting as a cofactor, NADPH protects DHFR from degradation in vitro and in a cellular context (5-8). DHFR protein binds to its own mRNA to self-regulate translation (9-12). The enzymatic activity and subcellular localization of DHFR are at least partially regulated by post-translational modification. Mono-ubiquitination of DHFR by MDM2 reduces the activity of DHFR (13). DHFR is primarily cytoplasmic, but it has been shown to translocate to the nucleus in a SUMO-dependent manner (14). Due to its role in nucleotide biosynthesis, DHFR has long been a therapeutic target for the treatment of various cancers and infectious diseases (15, 16).

References

  1. Bertino, J.R. et al. (1965) Biochemistry 4:839.
  2. Schweitzer, B.I. et al. (1990) FASEB J. 4:2441.
  3. Fierke, C.A. et al. (1987) Biochemistry 26:4085.
  4. Abali, E.E. et al. (2008) Vitam. Horm. 79:267.
  5. Perkins, J.P. et al. (1967) J. Biol. Chem. 242:4771.
  6. Wallace, L.A. and C. Robert Matthews (2002) J. Mol. Biol. 315:193.
  7. Ainavarapu, S.R. et al. (2005) Biophys. J. 89:3337.
  8. Hsieh, Y.C. et al. (2013) Mol. Pharmacol. 83:339.
  9. Chu, E. et al. (1993) Biochemistry 32:4756.
  10. Ercikan, E. et al. (1993) Adv. Exp. Med. Biol. 338:537.
  11. Ercikan-Abali, E.A. et al. (1997) Biochemistry 36:12317.
  12. Tai, N. et al. (2004) Biochem. J. 378:999.
  13. Maguire, M. et al. (2008) Cancer Res. 68:3232.
  14. Anderson, D.D. et al. (2007) Clin. Chem. Lab. Med. 45:1760.
  15. Goldman, I.D. et al. (2010) Curr. Opin. Investig. Drugs 11:1409.
  16. Sharma, M. and P.M. Chauhan (2012) Future Med. Chem. 4:1335.

Alternate Names

DHFR, DHFRP1, DYR

Entrez Gene IDs

1719 (Human); 13361 (Mouse); 24312 (Rat)

Gene Symbol

DHFR

UniProt

P00374 (Human)

Additional Dihydrofolate Reductase/DHFR Products

Product Documents for Recombinant Human DHFR Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Download SDS

Product Specific Notices for Recombinant Human DHFR Protein, CF

For research use only

Related Research Areas

Redox Enzymes

Citations for Recombinant Human DHFR Protein, CF

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Protocols

View specific protocols for Recombinant Human DHFR Protein, CF (8456-DR):

Materials
  • Assay Buffer: 50 mM MES, 25 mM Tris, 100 mM NaCl, 25 mM Ethanolamine, 2 mM DTT
  • Recombinant Dihydrofolate Reductase/DHFR (rhDHFR) (Catalog # 8456-DR)
  • beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog # N7505), 10 mM stock in deionized water
  • Dihydrofolic acid (DHF)  (Sigma, Catalog # D7006), 10 mM stock in Assay Buffer + 4.5 mM NaOH
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhDHFR to 1 μg/mL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 0.2 mM DHF and 0.25 mM beta -NADPH in Assay Buffer.
  3. Load 50 μL of 1 μg/mL rhDHFR into a plate, and start the reaction by adding 50 μL of Substrate Mixture.  Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  4. Read at an absorbance of 339 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol x (-1)
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank
**Using the extinction coefficient 6220 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD. Per Well:

  • rhDHFR: 0.05 μg
  • DHF: 0.1 mM
  • beta -NADPH: 0.125 mM

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