Recombinant Mouse CHST5 Protein, CF

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

Recombinant Mouse CHST5 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to N-acetyl-D-glucosamine. The specific activity is >300 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse Carbohydrate Sulfotransferase 5/CHST5 protein
  Ser27-Ser395, with an N-terminal 6-His tag
  Accession # NP_064334
• Accession #: NP_064334
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Chst5
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 42 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: 57 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5
  • Recombinant Mouse Carbohydrate Sulfotransferase 5/CHST5 (rmCHST5) (Catalog # 5210-ST)
  • 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (PAPS) (Catalog # ES019)
  • N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog #1079), 1 M 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, 600 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rmCHST5 to 4 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 4 µg/mL rmCHST5 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:
  • rmCHST5: 0.1 μg
  • Coupling Phosphatase 3: 0.5 μg
  • PAPS: 0.2 mM
  • GlcNAc: 300 mM

Notes

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

Alternate Names for Recombinant Mouse CHST5 Protein, CF

• carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5
• Carbohydrate Sulfotransferase 5
• CHST5
• GlcNAc6ST3
• gn6st-3
• GST4-alpha
• hIGn6ST
• I-GlcNAc6ST

Background

The CHST family is comprised of 14 genes in both the human and mouse genomes. All members of this family are Golgi-localized type II membrane proteins. Only the luminal and enzymatic domain is expressed in each of our recombinant CHST proteins. These enzymes transfer sulfate (i.e., sulfonate) onto the 6-O or 4‑O positions of GalNAc, Gal and GlcNAc residues on glycoproteins, proteoglycans and glycolipids (1). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (2). CHST5, also designated as I-GlcNAc6ST, GlcNAc6ST-3, and GST-4 alpha (3, 4), has a marked preference for sulfating O-linked sugars of mucin type of acceptors (3) and catalyzes the transfer of sulfate to position 6 of non-reducing GlcNAc residues on keratan sulfate (5, 6). The amino acid sequence for mouse CHST5 has higher identity to that of human CHST6 (83%) than to human CHST5 (76%). Since there is no CHST6 found in the mouse genome it is possible that mouse CHST5 plays a similar biological role to the human CHST6 (6).The activity of the recombinant CHST6 has been measured with a phosphatase-coupled sulfotransferase assay (7).

1. Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
2. Bowman, K. G. and C. R. Bertozzi (1999) Chem. Biol. 5:447.
3. Lee, J. K. et al. (2002) Glycobiology 13:245.
4. Lee, J. K. et al. (1999) Biochem. Biophys. Res. Commun. 263: 543.
5. Akama, T. O. et al. (2001) J. Biol. Chem. 276:16271.
6. Akama, T. O. et al. (2002) J. Biol. Chem. 277:42505.
7. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Bioinformatics

• Gene Symbol: Chst5
• Uniprot:
  • NP_064334()