Recombinant Human DHFR Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human DHFR Protein, CF Summary

• Details of Functionality: Measured by the reduction of dihydrofolic acid (DHF). The specific activity is >5,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Dihydrofolate Reductase/DHFR protein
  Met1-Asp187, with a C-terminal 6-His tag
• Accession #: P00374
• N-terminal Sequence: Met1 & Val2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: DHFR
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 22 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 23 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human DHFR Protein, CF

• DHFR
• DHFRP1
• Dihydrofolate Reductase
• DYR
• EC 1.5.1.3

Background

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).

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.

Bioinformatics

• Gene Symbol: DHFR
• Uniprot:
  • P00374(Human)