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Recombinant Human PON1 His-tag Fc Chimera Protein, CF

Catalog #: 10175-PO Datasheet
Catalog # Availability Size / Price Qty
10175-PO-020
Recombinant Human PON1 His-tag Fc Chimera Protein Enzyme Activity
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Recombinant Human PON1 His-tag Fc Chimera Protein, CF Summary

Product Specifications

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.
Activity
Measured by its ability to hydrolyze phenyl acetate. The specific activity is >5000 pmol/min/μg, as measured under the described conditions.
Source
Human embryonic kidney cell, HEK293-derived human PON1 protein
Leu16-Leu355
with an N-terminal 6-His tag and a C-terminal Fc tag
Accession #
P27169
N-terminal Sequence
Analysis
His
Predicted Molecular Mass
66 kDa
SDS-PAGE
69-79 kDa, under reducing conditions

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10175-PO

Product Datasheet

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

10175-PO

Formulation Supplied as a 0.2 μm filtered solution in Tris, CaCl2, NaCl, and Glycerol.
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.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 5 mM CaCl2, pH 8.0
  • Deionized Water
  • Recombinant Human PON1 His-tag Fc Chimera (rhPON1) (Catalog 10175-PO)
  • Substrate: Phenyl Acetate (7.88 M) (Sigma, Catalog # 108723)
  • UV Plate (Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPON1 to 10 µg/mL in Assay Buffer.
  2. Dilute Phenyl Acetate to 40 mM in deionized water.
  3. Load 50 µL of 10 µg/mL rhPON1 in a plate, and start the reaction by adding 50 µL of Substrate.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate.
  4. Read plate at a wavelength of 270 nm (absorbance) 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 1310 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD

Per Well:
  • rhPON1: 0.5 µg
  • Phenyl Acetate: 20 mM

Scientific Data

Enzyme Activity Recombinant Human PON1 His-tag Fc Chimera Protein Enzyme Activity View Larger

Recombinant Human PON-1 His-tag Fc Chimera (Catalog # 10175-PO) is measured by its ability to hydrolyze phenyl acetate. The activity (orange) is over 3-fold higher than the competitor's PON-1 (green).

SDS-PAGE Recombinant Human PON1 His-tag Fc Chimera Protein SDS-PAGE View Larger

2 μg/lane of Recombinant Human PON1 His-tag Fc Chimera was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® blue staining, showing a band at ~74 kDa under reducing conditions.

Background: PON1

Serum paraoxonase 1 (PON-1) is a member of the paraoxonase family which has three members: PON1, PON2, PON3.  PON-1 is a lactonase (1,2) but has significant promiscuity (2,3) that allows hydrolysis of a variety of substrates including organophosphate triesters (including many pesticides), arylesters, cyclic carbamates, glucuronides and consequently also pharmaceuticals such as statins. PON-1 is a calcium-dependent, secreted, 43 kDa protein with a 6-bladed propeller structure and an active site gorge containing a His-His catalytic dyad (4).  It is a homodimer and retains its hydrophobic signal sequence in the N-terminal region which enables its association with HDL, resulting in its localization predominantly in the plasma (5,6). Low serum PON1 and dysfunctional HDL is associated with many diseases with an inflammatory component including diabetes mellitus (7,8), rheumatoid arthritis, hepatic and renal diseases, psoriasis, and macular degeneration (9,10). PON1 shows a variety of atheroprotective properties (6,11,12) by metabolizing inflammatory lipid peroxides (13). PON-1 activity is low in coronary heart disease patients (14) and contributes to an increased risk of a major adverse cardiovascular event (15). PON-1 also protects against toxicity of organophosphorus compounds used as pesticides (16,17) thought to increase risk of neurodegenerative disorders such as Parkinson's (18) and Amyotrophic Lateral Sclerosis (19).

References
  1. Dragonov, D. I. et al. (2005) Lipid Res. 46:1239.
  2. Khersonsky, O. et al. (2005) Biochemistry. 44:6371.
  3. Ben-David, M. et al. (2012) J. Mol. Biol. 418:181.
  4. Harel, M. et al. (2004) Nature Struct. Mol. Biol. 11:412.
  5. Mackness, B. et al. (1998) Gen. Pharmac. 31:329.
  6. Aviram, M. et al. (2004) Free Radic. Biol. Med. 37:1304.
  7. Rozenberg, O. et al. (2008) Free Radic. Biol. Med. 44:1951.
  8. Koren-Gluzer, M. et al. (2011) Atherosclerosis. 219:532.
  9. Goswami, N. et al. (2009) Clin. Chim. Acta. 410:1.
  10. Mahrooz, A. (2016) Curr. Clin. Pharmacol. 11:259.
  11. Berrougui, H. et al. (2012) Free Radic. Biol. Med. 52:1372.
  12. Aharoni, S. et al. (2013) Atherosclerosis 228:353.
  13. Tavori, H. (2011) Free Rad. Biol. Med. 51:234.
  14. Wang, M. et al. (2012) DNA Cell. Biol. 31:975.
  15. Tang, W. H. W. et al. (2012) Arterioscler. Thromb. Vasc. Biol. 32:2803.
  16. Li, W. F. et al. (2000) Pharmacogenetics. 10:767.
  17. Shih, D. M. et al. (1998) Nature. 394:284.
  18. Ahmed, H. et al. (2017) Biomed. Pharmacother. 90:638.
  19. Gagliardi, S. et al. (2013) Neurotox. Res. 23:370.
Long Name
Paraoxonase 1
Entrez Gene IDs
5444 (Human); 18979 (Mouse); 84024 (Rat)
Alternate Names
A-esterase 1; Aromatic esterase 1; arylesterase B-type; EC 3.1.1.2; EC 3.1.1.81; EC 3.1.8.1; ESA; ESAesterase A; K-45; MVCD5; paraoxonase 1; PON 1; PON1; PONparaoxonase B-type; serum aryldiakylphosphatase; Serum aryldialkylphosphatase 1; serum paraoxonase/arylesterase 1

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