Recombinant Human PHEX Protein, CF Summary
Product Specifications
Gln42-Trp749, with an N-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
8090-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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 Procedure
- Assay Buffer: 50 mM MES, 250 mM NaCl, pH 6.0
- Recombinant Human PHEX (rhPHEX) (Catalog # 8090-ZN)
- Substrate: Mca-YVADAPK(Dnp)-OH (Catalog # ES007)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhPHEX to 4 ng/µL in Assay Buffer.
- Dilute Substrate to 100 µM in Assay Buffer.
- Load 50 µL of 4 ng/µL rhPHEX into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhPHEX: 0.2 µg
- Substrate: 50 µM
Reconstitution Calculator
Background: PHEX
PHEX (PHosphate-regulating gene with homology to Endopeptidases on the X-chromosome; also known as metallopeptidase homolog PEX, vitamin D resistant hypophosphatemic rickets protein and X-linked hypophosphatemia protein/HYP) is a monomeric, zinc-dependent transmembrane member of the metallopeptidase M13 family of enzymes (1-6). Members of the M13 family are membrane-bound and characterized by the presence of a catalytic domain that consists of an ExxAG (or D) endopeptidase sequence, coupled to a canonical Zn-binding HExxH motif (1, 3). PHEX expression is limited to a small number of cell types, including mature osteoblasts (7, 8), osteocytes (8, 9), and odontoblasts (but not ameloblasts) (7, 8). PHEX appears to have a material effect on mineralization. Typically, mineralization occurs when inorganic phosphate interacts with calcium to form hydroxyapatite. These crystals are deposited on the surface of either collagen I or DMP1 in bone matrix, and serve as nucleation sites for additional mineral deposition (10, 11). At select timepoints, osteocytes will downregulate the rate of mineralization, principally to insure that space exists between cell processes and calcified matrix. This is accomplished through the secretion of SIBLING family molecules (OPN; MEPE) that possess RGD and ASARM (Acidic Serine-and-Aspartate-Rich Motif) sequences. When cathepsin-like molecules are available, SIBLING molecules are cleaved, releasing ASARMs. If phosphorylated, these ASARMs bind to naked nucleation sites and terminate the growth of mineral. PHEX has a high affinity for ASARMs, and will cleave these fragments into smaller fragments, thus protecting the nucleation centers and promoting mineralization (12-14). In addition, PHEX will also bind the ASARM sequence in full-length SIBLING molecules (such as MEPE), possibly protecting them from proteolysis (15, 16). This blocks the generation of ASARMs that serve as antagonists to mineralization (15). Human PHEX is a 90-100 kDa, 749 amino acid (aa) type II transmembrane glycoprotein (3, 8, 9, 17). It contains a 20 aa cytoplasmic segment and a 708 aa extracellular region (aa 42-749) that possesses a lengthy catalytic domain (aa 67-741). Multiple point mutations are noted that adversely affect PHEX activity. There is one potential isoform variant that shows a six aa substitution for aa 1-293. Full-length human and mouse PHEX share 96% aa sequence identity.
- Turner, A.J. et al. (2001) BioEssays 23:261.
- Francis, F. et al. (1995) Nat. Genet. 11:130.
- Lipman, M.L. et al. (1998) J. Biol. Chem. 273:13729.
- Grieff, M. et al. (1997) Biochem. Biophys. Res. Commun. 231:635.
- Beck, L. et al. (1997) J. Clin. Invest. 99:1200.
- Guo, R. & L.D. Quarles (1997) J. Bone Miner. Res. 12:1009.
- Ruchon, A.F. et al. (1998) J. Histochem. Cytochem. 46:459.
- Ruchon, A.F. et al. (2000) J. Bone Miner. Res. 15:1440.
- Liu, S. et al. (2002) J. Biol. Chem. 277:3686.
- Sapir-Koren, R. & G. Livshits (2011) IBMS BoneKEy 8:286.
- Gajjeraman, S. et al. (2007) J. Biol. Chem. 282:1193.
- Rowe, P.S. (2012) Cell Biochem. Funct. 30:355.
- Addison, W.N. et al. (2010) J. Bone Miner. Res. 25:695.
- Barros, N. et al. (2013) J. Bone Miner. Res. 28:688.
- Cavalli, L. et al. (2012) Clin. Cases Miner. Bone Metab. 9:9.
- Rowe, P.S.N. et al. (2005) Bone 36:33.
- Liu, S. et al. (2003) J. Biol. Chem. 278:37419.
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