Recombinant Human HS3ST4 Protein, CF

R&D Systems | Catalog # 6085-ST

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human HS3ST4 Protein (6085-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

NP_006031

Applications

Enzyme Activity
View all Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4 protein
Gly184-Lys456 with an N-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

N-terminal Sequence Analysis

HHHHHHGGAV

Predicted Molecular Mass

32 kDa

SDS-PAGE

35-43 kDa, reducing conditions

Activity

Measured by its ability to transfer sulfate from PAPS to heparin.The specific activity is >30 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

6085-ST
Formulation Supplied as a 0.2 μm filtered solution in Tris, 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.

Background: Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4

Heparan sulfate is a highly sulfated polysaccharide that can be found on the cell surface and within the extracellular matrix. It is typically 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 can be sulfated at the 2-O position by heparan sulfate 2‑O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at the N, 3-O, and 6-O positions by N‑deacetylase/N‑sulfotransferases (NDSTs), heparan sulfate 3‑O sulfotransferases (HS3STs) and heparan sulfate 6-O sulfotransferases (HS6STs) respectively. There are seven HS3STs in the human genome (3, 4). HS3ST4 and HS3ST2 are brain specific and may participate in HS-dependent neurobiologic events (5). HS3ST4 can generate tetrasulfated heparan sulfate disaccharide, the most highly sulfated sugar found in biological samples (6, 7), and may have a role in assisting HSV-1 entry and spread (8). HS3ST4 is a Golgi resident type II membrane protein and has the longest proline rich stem region among all HS3STs (3, 5). The activity of the recombinant human CHST1 is measured using a PAP-specific phosphatase-coupled sulfotransferase assay (9).

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. Shworak, N.W. et al. (1999) J. Biol. Chem. 274:5170.
  4. Xu, D. et al. (2005) Biochem. J. 386:451.
  5. Lawrence, R. et al. (2007) Matrix Biol. 26:442.
  6. Mochizuki, H. et al. (2003) J. Biol. Chem. 278:26780.
  7. Wu, Z.L. et al. (2004) J. Biol. Chem. 279:1861.
  8. Tiwari,V. et al. (2005) Biochem. Biophys. Res. Commun. 338:930.
  9. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Alternate Names

3-OST-4

Entrez Gene IDs

9951 (Human)

Gene Symbol

HS3ST4

UniProt

NP_006031

Additional Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4 Products

Product Documents for Recombinant Human HS3ST4 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human HS3ST4 Protein, CF

For research use only

Citations for Recombinant Human HS3ST4 Protein, CF

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Protocols

View specific protocols for Recombinant Human HS3ST4 Protein, CF (6085-ST):

Materials
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5
  • Recombinant Human HS3ST4 (rhHS3ST4) (Catalog # 6085-ST)
  • Donor Substrate: PAPS (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # ES019)
  • Acceptor Substrate: Heparin, 20 mg/mL stock in deionized water
  • Clear 96-well Plate (Catalog # DY990)
  • Plate Reader with Absorbance Read Capability
  1. Prepare 1X Assay Buffer by diluting 10X stock 10-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. Prepare a Reaction Mixture containing 0.4 mM PAPS, 4 mg/mL Heparin and 20 μg/mL Coupling Phosphatase 3 (supplied in kit) in 1X Assay Buffer.
  6. Dilute rhHS3ST4 to 20 µg/mL in 1X Assay Buffer.
  7. Load 25 µL of 20 µg/mL rhHS3ST4 into empty wells of the same plate as the curve. Include a Control containing 25 µL of 1X Assay Buffer.
  8. Add 25 µL of Reaction Mixture to 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.  Mix briefly.
  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:

  • rhHS3ST4: 0.5 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Heparin: 100 µg
  • PAPS: 0.2 mM

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