Recombinant Human Asparagine Synthetase/ASNS His Protein, CF

2 μg/lane of Recombinant Human Asparagine Synthetase/ASNS His-tag was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® blue staining, showing a band at ~62 kDa under reducing conditions.

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

Recombinant Human Asparagine Synthetase/ASNS His Protein, CF Summary

• Details of Functionality: Measured by its ability to produce diphosphate during the conversion of aspartate and glutamine to asparagine and glutamate. The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human Asparagine Synthetase/ASNS protein
  Cys2-Ala561
  with C-terminal 6-His tag
• Accession #: P08243-1
• N-terminal Sequence: Cys
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 65 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: 58-65 kDa, under 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, NaCl, TCEP and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 100 mM Tris, 100 mM NH₄Cl, 10 mM MgCl₂, pH 8.0
  • Recombinant Human Asparagine Synthetase/ASNS (rhASNS) (Catalog # 10193-AS)
  • Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
  • L-Aspartic Acid (Sigma, Catalog # A7219), 200 mM stock in deionized water
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 400 mM stock in deionized water
  • L-Glutamine (Sigma, Catalog # G8540), 200 mM stock in deionized water
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  2. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare reaction mixture containing 2 mM ATP, 8 mM L-Aspartic, 40 mM L-Glutamine and 8 µg/mL ryPPA1 in Assay Buffer.
  4. Dilute rhASNS to 16 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 16 µg/mL rhASNS into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  8. Seal plate and incubate at room temperature for 10 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 sealed plate 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) x 2)

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

  Per Reaction:
  • rhASNS: 0.4 µg
  • ryPPA1: 0.2 µg
  • ATP: 1 mM
  • L-Aspartic Acid: 4 mM
  • L-Glutamine: 20 mM

Notes

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

Alternate Names for Recombinant Human Asparagine Synthetase/ASNS His Protein, CF

• ASNS
• ASNSD
• asparagine synthetase (glutamine-hydrolyzing)
• asparagine synthetase [glutamine-hydrolyzing]
• asparagine synthetase
• Cell cycle control protein TS11
• EC 6.3.5.4
• Glutamine Dependent Asparagine Synthetase
• Glutamine-dependent asparagine synthetase
• TS11 Cell Cycle Control Protein
• TS11

Background

Asparagine Synthetase (ASNS) represents a single mammalian-expressed enzyme that catalyzes the synthesis of asparagine and glutamate from aspartate and glutamine in an ATP-dependent amidotransferase reaction (1). Although the name focuses attention on its function in asparagine synthesis, the reaction catalyzed by ASNS also impacts glutamine, aspartate and glutamate homeostasis. Human ASNS is a class II or N-terminal nucleophile glutamine amidotransferase (2) and is a cytosolic, 65 kDa ATP-dependent homodimer where each monomer is composed of two functional domains based on modeling: an N-terminal domain that contains the glutamine-binding pocket and the C-terminal domain that contains the ATP-binding site (3). Although ubiquitously expressed at low levels in many organs, expression is particularly high in the pancreas (4).  Several mutations in the ASNS gene which are suspected to result in decreased activity cause Asparagine Synthetase Deficiency (ASD) (5). ASD has been characterized as a neurological disorder with severe impacts on psychomotor development and mortality at a young age supporting involvement of asparagine in neural development (6). As deficiency of ASNS leads to extracellular asparagine dependence, the brain is highly susceptible to deficiency due to the blood brain barrier limitation of available amino acids (7). Interest in targeting asparagine metabolism in cancer is based on the observation that unlike most normal cells, in acute lymphatic leukemia (ALL) there is little or no detectable ASNS nor upregulation during substrate deprivation in leukemic lymphoblasts making the transformed cells specifically sensitive to extracellular asparagine depletion (5, 8-10). Lower ASNS levels correlates with reduced proliferative capacity while subsequent supplementation of asparagine led to increased cell survival (11). Treatment with bacterial asparaginase as a component in combinatorial chemotherapy depletes plasma asparagine levels and results in starving leukemia cells in ALL to prevent growth (12) and can be applied more broadly to solid tumors where ASNS expression levels correlate with asparaginase sensitivity (10, 13-15).

1. Richards, N. G. J. and Schuster, S.M. (1998) Adv. Enzymol. 72:145.
2. Zalkin, H. and J. L. Smith (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72:87.
3. Larsen, T. M. et al. (1999) Biochemistry. 38:16146
4. Milman, H. A. and D. A. Cooney (1974) Biochem. J. 142:27.
5. Lomelino, C. L. et al. (2017) J. Biol. Chem. 292:19952.
6. Ruzzo, E. K. et al. (2013) Neuron. 80:429.
7. Hawkins, R. A. et al. (2006) J. Nutr. 136:218S.
8. Haskell, C. M. et al. (1969) Biochem. Pharmacol. 18:2578.
9. Aslanian, A. M. et al. (2001) Biochem. J. 357:321.
10. Su, N. et al. (2008) Pediatr. Blood Cancer. 50:274.
11. Ye, J. et al. (2010) EMBO J. 29:2082.
12. Pieters, R. et al. (2011) Cancer 117:238.
13. Lorenzi, P. L. et al. (2008) Mol. Cancer Ther. 7:3123.
14. Dufour, E. et al. (2012) Pancreas. 41:940.
15. Du, F. et al. (2019) Cell Death Dis. 10:239.

Bioinformatics

• Uniprot:
  • P08243-1()