Recombinant Human HPRT His-tag Protein, CF

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2 μg/lane of Recombinant Human HPRT His-tag (Catalog # 10314-HP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 27-29 ...read more
Recombinant Human HPRT Hig-tag (Catalog # 10314-HP) is measured by its ability to produce GMP from guanine and phosphoribosyl pyrophosphate. The activity (orange) is over 2-fold higher than the competitor's HPRT (blue).

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human HPRT His-tag Protein, CF Summary

Details of Functionality
Measured by its ability to produce GMP from guanine and phosphoribosyl pyrophosphate. The specific activity  is >9000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human HPRT protein
Ala2-Ala218
with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Met
Protein/Peptide Type
Recombinant Enzymes
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
25 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
27-29 kDa, under 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 and TCEP.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  •  Assay Buffer: 0.1 M Tris, 100 mM MgCl2, pH 8.5
  • Recombinant Human HPRT His-tag (rhHPRT) (Catalog # 10314-HP)
  • 5-phospho-D-ribose 1-diphosphate pentasodium salt (PRPP) (Sigma, Catalog # P8296), 50 mM stock in deionize water
  • Guanine (Sigma, Catalog # G11950), 2 mM stock in deionized water with addition of 10 mM NaOH (or until pH of ~12) to solubilize
  • UV Plate (Corning, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhHPRT to 0.4 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 2 mM PRPP and 120 µM Guanine in Assay Buffer.
  3. Load 50 µL of 0.4 µg/mL rhHPRT into plate, and start the reaction by adding 50 µL of Substrate Mixture.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read plate at an absorbance of 257 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
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

  

 *Adjusted for Substrate Blank
 **Using the extinction coefficient 5817 M-1cm-1
 ***Using the path correction 0.32 cm
 Note: the output of many spectrophotometers is in mOD

Per Well:
  • rhHPRT: 0.020 µg
  • PRPP: 1 mM
  • Guanine: 60 µM

Notes

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

Alternate Names for Recombinant Human HPRT His-tag Protein, CF

  • EC 2.4.2.8
  • HGPRT
  • HGPRT1
  • HGPRTHPRTHGPRTase
  • HPRT
  • HPRT1
  • hypoxanthine phosphoribosyltransferase 1
  • hypoxanthine-guanine phosphoribosyltransferase

Background

Hypoxanthine-guanine phosphoribosyltransferase (HPRT), a member of the phosphoribosyltransferase family, is a magnesium-dependent, cytoplasmic, ubiquitously-expressed (1) salvage enzyme involved in the primary pathway utilized for purine synthesis (2, 3). It catalyzes the transfer of phosphoribose from phosphoribosyl pyrophosphate to hypoxanthine and guanine bases to form inosine and guanine, respectively. Human HPRT forms a homotetramer of identical subunits that form dimer pairs (4, 5). Each 49 kDa subunit is 217 amino acids and consists of a core domain containing the active site and a hood domain (4, 5). HPRT deficiency in the salvage pathway leads to overproduction of purine, indicating it may play a role in regulation of purine production (6). Deficiency from multiple characterized single point mutations (7,8) causes hyperuricemia resulting in gout-like symptoms of Kelley-Seegmiller syndrome or if completely lacking in HPRT, a severe X-linked hereditary disorder, Lesch-Nyhan Disease, that is also characterized to cause significant neurological impairment (3, 6) by affecting multiple signaling pathways (9) and through dysregulation of cellular functions including proliferation, RNA metabolism, DNA replication and protein synthesis (3, 10). Given its ability to modulate cellular functions through purine regulation, HPRT is implicated to play a role in cancer. Significant correlation between HPRT mutations and increased cancer risk have been reported (11, 12). HPRT has been found surface-expressed in cancer (13) and thus has been proposed as a potential marker and therapeutic target (12).
  1. Melton, D. W. et al. (1986) Cell 44:319.
  2. Stout, J. T. et al. (1985) Ann. Rev. Genet. 19:127.
  3. Fasullo, M. and L. Endres. (2015) Int. J. Mol. Sci. 16:9431.
  4. Eads, J. C. et al. (1994) Cell 78:325.
  5. Keough, D.T. et al. (2005) J. Mol. Biol. 351:170.
  6. Garcia-Gil, M. et al. (2018) Int. J. Mol. Sci. 19: E3598.
  7. Torres, R. J. and J.G. Puig (2007) Orphanet J. Rare Dis. 2:48.
  8. Zoref-Shani, E. et al. (2000) BBA Mol. Basic Dis. 1500:197.
  9. Guibinga, G. H. et al. (2014) PLoS ONE 9:e96575.
  10. Kang. T. H. et al. (2013 PLoS ONE 8:e74967.
  11. Townsend, M. H. et al. (2017) Cancer Clin. Oncol. 6:19.
  12. Townsend, M. H. et al. (2018) Med. Oncol. 35:89.
  13. Townsend, M. H. et al. (2017) OncoTargets Ther. 10:1921.

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