Recombinant Human TPST1 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Human TPST1 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer sulfate from PAPS to PSGL-1 peptide (Gln-Ala-Thr-Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro-Glu-Thr) The specific activity is >5 pmol/min/μg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Tyrosylprotein Sulfotransferase 1/TPST1 protein
Gln26-Glu370, with an N-terminal 6-His tag
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
TPST1
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

Theoretical MW
40 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
40-50 kDa, reducing conditions
Publications
Read Publication using
5468-ST in the following applications:

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
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 25 mM MES, 0.5% (v/v) Triton® X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, pH 7.0
  • Gel Running Buffer: 40 mM Tris, 1 mM EDTA, adjust to pH 8.0 with acetic acid
  • Recombinant Human Tyrosylprotein Sulfotransferase 1/TPST1  (rhTPST1) (Catalog # 5468-ST)
  • 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
  • PSGL-1 Peptide (Gln-Ala-Thr-Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro-Glu-Thr), 5 mM in Assay Buffer
  • PAPS (Sigma, Catalog # A1651), 1 mM stock solution in 5% ethanol, 95% deionized water
  • PAP35S (prepared in-house) (7-9), ~1 μM in 5% ethanol, 95% deionized water
  • Gel loading buffer: 0.15 M Tris, 20.8 mM SDS, 1.15 M Glycine, 174 µM Bromophenol Blue, 30% Glycerol
  • Blotting paper (Fisher Scientific, Catalog # 05-714-4)
  • Gel dryer
  • Glogos® II autorad markers (Stratagene, Catalog # 420202) or equiv.
  • Blue sensitive medical X-ray film
  • X-ray film cassette
  • Film developer (Konica SRX-101A Medical Film Processor) or equiv.
  • Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equiv.
  • Liquid scintillation fluid (Beckman Coulter, Catalog # 141349) or equiv.
  1. Dilute rhTPST1 to 100, 50, 25 and 12.5 µg/mL in Assay Buffer.
  2. Combine 10 µL PAPS, 10 µL PAP35S, 35 µL PSGL-1 peptide, and 85 µL deionized water (rxn mix sufficient for ~9 rxns).
  3. Combine 15 µL enzyme at each dil. with 15 µL rxn mix. Include a control containing 15 µL Assay Buffer and 15 µL rxn mix.
  4. Incubate at 37 °C for 20 minutes
  5. Add 15 µL gel loading buffer to each rxn. Mix.
  6. Load 30 µL of each rxn per lane on a gel. Leave empty lanes between samples.
  7. Run at 200 V for 25 minutes
  8. Transfer gel onto blotting paper and dry with gel dryer for 1 hr or until fully dry.
  9. Affix two autorad markers to the blotting paper next to the dried gel.
  10. In a darkroom expose dried gel to X-ray film by enclosing overnight in film cassette.
  11. After developing, use autorad markers to align X-ray film with gel.
  12. Determine where labeled product (slowest), unreacted PAP35S (intermediate), and free sulfate (fastest) migrated on the gel. For the control, identify the empty region where the product would have migrated if any had been produced.
  13. Cut out regions containing product and unreacted PAP35S.
  14. Place each region into a separate liquid scintillation vial.
  15. Add 5 mL liquid scintillation fluid to each vial.
  16. Use a liquid scintillation counter to count the amount of 35S in each vial.
  17. Calculate Activity for each rxn using the following equation. Plot Activity vs. μg per reaction, fit to linear regression. The slope of the fit equals the Specific Activity (pmol/min/μg):

     Activity (pmol/min) =

S (pmol) x Ci (counts)
Ct (counts) x time (min)
S = applied donor substrate Ci = incorporated radioisotope (product) Ct = total applied radioisotope (product plus unreacted PAP35S)
Per Reaction:
  • PAPS: 1000 pmol (pmol of PAP35S in the assay is negligible)
  • rhTPST1: 1.5, 0.75, 0.375 and 0.1875 μg

Notes

Triton is a registered trademark of Union Carbide Corp. Glogos is registered trademark of Stratagene.

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

Alternate Names for Recombinant Human TPST1 Protein, CF

  • EC 2.8.2.20
  • protein-tyrosine sulfotransferase 1
  • TPST1
  • Tyrosylprotein Sulfotransferase 1
  • tyrosylprotein sulfotransferase 1TPST-1
  • tyrosylprotein sulfotransferase-1

Background

Tyrosine O-sulfation is a posttranslational modification of proteins in all multicellular organisms. This reaction is mediated by a Golgi enzyme activity called Tyrosylprotein Sulfotransferase (TPST) that transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to tyrosine residues within acidic motifs of polypeptides to form tyrosine O-sulfate ester (1). More than 60 proteins have been identified to be tyrosine sulfated, for some of which the functions have been identified. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P-selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P-selectin on activated endothelium (2, 3). Tyrosine sulfation of chemokine receptors CCR5 and CXCR4 have been reported to facilitate HIV-1 entry of target cells (4, 5). Two TPSTs with overlapping substrate specificities are found in the human genome. TPST1 is widely expressed in all major tissues. The TPST1 knock-out mice showed phenotype of reduced body weight and increased postimplantation fetal death (6).

  1. Ouyang, Y. et al. (1998) Proc. Natl. Acad. Sci. USA 95:2896.
  2. Danan, L.M. et al. (2008) J. Am. Soc. Mass. Spectrom. 19:1459.
  3. Somers, W.S. et al. (2000) Cell 103:467.
  4. Choe, H. et al. (2003) Cell 114:161.
  5. Seibert, C. et al. (2008) Biochemistry 47:11251.
  6. Ouyang, Y.B. et al. (2002) J. Biol. Chem. 277:23781.
  7. Robbins, P.W. (1962) Methods in Enzymology, Vol. V, Academic Press, Inc., New York, 964.
  8. MacRae, I.J. et al. (2000) Biochemistry 39:1613.
  9. Wu, Z.L. et al. (2002) FASEB J. 16:539.

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Publications for Tyrosylprotein Sulfotransferase 1/TPST1 (5468-ST)(1)

We have publications tested in 1 confirmed species: N/A.

We have publications tested in 1 application: Enzyme Assay.


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Bioinformatics

Gene Symbol TPST1
Uniprot