Recombinant Human HPRT His-tag Protein, CF Summary
with an N-terminal Met and 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and TCEP.|
|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.
- 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
- Dilute rhHPRT to 0.4 µg/mL in Assay Buffer.
- Prepare Substrate Mixture containing 2 mM PRPP and 120 µM Guanine in Assay Buffer.
- 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.
- Read plate at an absorbance of 257 nm in kinetic mode for 5 minutes.
- 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
- rhHPRT: 0.020 µg
- PRPP: 1 mM
- Guanine: 60 µM
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).
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 kDa under reducing conditions.
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).
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- Garcia-Gil, M. et al. (2018) Int. J. Mol. Sci. 19: E3598.
- Torres, R. J. and J.G. Puig (2007) Orphanet J. Rare Dis. 2:48.
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- Guibinga, G. H. et al. (2014) PLoS ONE 9:e96575.
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- Townsend, M. H. et al. (2017) Cancer Clin. Oncol. 6:19.
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- Townsend, M. H. et al. (2017) OncoTargets Ther. 10:1921.
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