Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

R&D Systems | Catalog # 5057-ST

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1 (5057-ST)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

AAY14736

Applications

Enzyme Activity
View all Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1 protein
Pro28-Trp401, with an N-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

His

Predicted Molecular Mass

45 kDa

SDS-PAGE

56-60 kDa, reducing conditions

Activity

Measured by its ability to transfer sulfate from PAPS to heparin.
The specific activity is >10 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

5057-ST
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA.
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.

Background: Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1

Heparan Sulfate is a highly sulfated polysaccharide that can be found on cell surface and extracellular matrix. It is usually covalently attached to a protein core as the glycan component of a proteoglycan. Heparan Sulfate interacts with a variety of proteins, such as growth factors, protease inhibitors, cytokines, lipoprotein lipase and viral envelope proteins, thus plays roles from cell growth, cell differentiation, cell motility, blood coagulation, lipid metabolism to viral infection (1, 2). Heparan Sulfate consists of repeating residues of uronic acid and N‑acetylglucosamine. The uronic acid residues can be sulfated at 2-O position by Heparan Sulfate 2‑O‑Sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at N-, 3-O, and 6-O positions by N‑deacetylase/N‑sulfotransferases (NDSTs), Heparan Sulfate 3-O and 6-O sulfotransferases (HS3STs and HS6STs) respectively. However, the reactions catalyzed by these sulfotransferases are normally incomplete on the whole chain of Heparan Sulfate. As a result, Heparan Sulfate displays enormous sequence diversity that allows it to interact with a wide spectrum of proteins differently. Among three HS6STs, HS6ST1 is the first to be cloned (3). Mice deficient of the HS6ST1 homologue gene showed embryonic lethality (4).The enzymatic activity of the recombinant human HS6ST1 is measured using a phosphatase-coupled assay (5).

References

  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. Habuchi, H et al. (1998) J. Biol. Chem. 273:9208.
  4. Habuchi, H et al. (2007) J. Biol. Chem. 282:15578.
  5. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Alternate Names

DKFZp547H098, EC 2.8.2, EC 2.8.2.-, FLJ25392, heparan sulfate 6-O-sulfotransferase 1, heparan-sulfate 6-sulfotransferase, HS6ST-1, HS6STheparan-sulfate 6-O-sulfotransferase 1, MGC116899, MGC116901

Entrez Gene IDs

9394 (Human); 50785 (Mouse); 316325 (Rat)

Gene Symbol

HS6ST1

UniProt

AAY14736

Additional Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1 Products

Product Documents for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

For research use only

Citations for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

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Protocols

View specific protocols for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF (5057-ST):

Materials
  • Assay Buffer: 50 mM Tris, 15 mM MgCl2, pH 7.5
  • Recombinant Human Heparan Sulfate 6‑O‑Sulfotransferase 1/HS6ST1 (rhHS6ST1) (Catalog # 5057-ST)
  • Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
  • Acceptor Substrate: Heparin, 20 mg/mL stock in deionized water
  • Sulfotransferase Activity Kit (Catalog # EA003)
  • Clear 96-well Plate (Costar, Catalog # 92592)
  • Plate Reader with Absorbance Read Capability
  1. Create Assay Buffer by Diluting 10X Phosphatase Buffer 3 (supplied in kit) to 1X in deionized water.
  2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  3. Continue 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.
  4. Prepare a reaction mixture containing 0.4 mM PAPS, 2 mg/mL Heparin, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer. *Note: Incorporate Coupling Phoshphatase 3 into reaction mixture right before the mixture is added to the plate in order to avoid unnecessary hydrolysis of PAPS.
  5. Dilute rhHS6ST1 to 60 µg/mL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 25 µL of the 60 µg/mL rhHS6ST1 into the plate. Include a control containing 25 µL of Assay Buffer.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  9. Seal plate and incubate at 37 °C for 30 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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:

  • rhHS6ST1: 1.5 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Heparin: 50 μg (1 mg/mL)
  • PAPS: 0.2 mM

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