Recombinant Human HS2ST1 Protein, CF

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

Recombinant Human HS2ST1 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >75 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 2-O-Sulfotransferase 1/HS2ST1 protein
  Met59-Asn356, with an N-terminal 6-His tag
• Accession #: Q7LGA3
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HS2ST1
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 36 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: 38-40 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer (provided in kit): 50 mM Tris, 15 mM MgCl₂, pH 7.5
  • Recombinant Human Heparan Sulfate 2-O-Sulfotransferase 1/HS2ST1 (rhHS2ST1) (Catalog # 6335-ST)
  • 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (Catalog # ES019)
  • Heparan sulfate (Celsus Laboratories, Catalog # HO-03101), 50 mg/mL stock in deionized water
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare standard curve by performing six one‑half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare reaction mixture containing 0.4 mM PAPS, 6 mg/mL Heparan sulfate, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rhHS2ST1 to 20 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 20 µg/mL rhHS2ST1 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

  Per Reaction:
  • rhHS2ST1: 0.5 μg
  • Coupling Phosphatase 3: 0.5 μg
  • PAPS: 0.2 mM
  • Heparan sulfate: 150 μg

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human HS2ST1 Protein, CF

• 2OST
• 2-O-sulfotransferase
• EC 2.8.2
• EC 2.8.2.-
• FLJ11317
• heparan sulfate 2-O-sulfotransferase 1
• HS2ST
• HS2ST1
• KIAA0448dJ604K5.2
• MGC131986

Background

Heparan sulfate is a highly sulfated polysaccharide found on the cell surface and within the extracellular matrix. Typically, it is covalently attached to the protein core of proteoglycans, such as syndecans and glypicans. Heparin, on the other hand, can be considered as a highly sulfated version of heparan sulfate that is predominantly found in mast cells. Both heparin and heparan sulfate contain disaccharide repeats of uronic acid and N‑acetylglucosamine and are modified by the same sulfotransferases (1, 2). The uronic acid residues are either glucuronic acid or iduronic acid and maybe sulfated at the 2-O position by heparan sulfate 2‑O sulfotransferase 1 (HS2ST1) (3, 4). HS2ST1 physically interacts in the Golgi apparatus with glucuronyl c5-epimerase (5), which catalyzes the conversion of glucuronic acid to iduronic acid (6). As a consequence, 2-O sulfation predominantly occurs on iduronic acids naturally and overexpression of HS2ST1 alone causes an increase in 2-O sulfation on glucuronic acid (7). The enzyme activity of the recombinant human HS2ST1 was measured using a PAP-specific phosphatase-coupled sulfotransferase assay (8).

1. Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
2. Esko, J.D. and Selleck, S.B. (2002) Annu. Rev. Biochem. 71:435.
3. Kobayashi, M. et al. (1996) J. Biol. Chem. 271:7645.
4. Bullock, S.L. et al. (1998) Genes & Development 12:1894.
5. Li, J.P. et al. (2001) J. Biol. Chem. 276:20069.
6. Pinhal, M.A.S et al. (2001) Proc. Natl. Acad. Sci. USA. 98:12984.
7. Rong, J. et al. (2000) Biochem. J. 346:463.
8. Prather, B. et al. (2012) Anal Biochem. 423:86.

Publications for Heparan Sulfate 2-O-Sulfotransferase 1/HS2ST1 (6335-ST)(1)

Publication using 6335-ST

• Qin Y, Ke J, Gu X, Fang J, Wang W, Cong Q, Li J, Tan J, Brunzelle J, Zhang C, Jiang Y, Melcher K, Li J, Xu H, Ding K Structural and functional study of D-glucuronyl C5-epimerase. J Biol Chem, 2015;290(8):4620-30. 2015 [PMID: 25568314] (Bioassay, Zebrafish)

Bioinformatics

• Gene Symbol: HS2ST1
• Uniprot:
  • Q7LGA3()