Recombinant Mouse alpha-L-Iduronidase/IDUA Protein, CF

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

Recombinant Mouse alpha-L-Iduronidase/IDUA Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl alpha -L-iduronide. The specific activity is >7,500 pmol/min/ug, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse alpha-L-Iduronidase/IDUA protein
  Glu17-Ser634, with a C-terminal 6-His tag
• Accession #: P48441
• N-terminal Sequence: Glu17
• 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: 70 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: 83-95 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 Sodium Acetate, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM NaOAc, 150 mM NaCl, 0.02% Brij-35 (w/v), pH 3.5
  • Developing Buffer: 0.1 M Tris, pH 9.0
  • Recombinant Mouse alpha -L-Iduronidase/IDUA (rmIDUA) (Catalog # 9348-GH)
  • Substrate: 4-methylumberlliferyl-alpha -L-Iduronide (Glycosynth, Catalog # 44076), 20 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmIDUA to 0.2 µg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.
  2. Dilute Substrate to 200 µM in Assay Buffer.
  3. Combine equal volumes of 0.2 µg/mL rmIDUA and 200 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.
  4. Incubate for 10 minutes at room temperature.
  5. Dilute mixtures to 0.005 µg/mL rmIDUA in Developing Buffer.
  6. In a plate load 100 µL of diluted mixtures.
  7. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in endpoint mode.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Blank.
  **Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).

  Per Well:
  • rmIDUA: 0.0005 µg
  • Substrate: 5 µM

Notes

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

Alternate Names for Recombinant Mouse alpha-L-Iduronidase/IDUA Protein, CF

• alphaLIduronidase
• alpha-L-Iduronidase
• IDA
• IDUA
• MPS1
• MPSI

Background

alpha -L-Iduronidase encoded by the IDUA gene is an important enzyme required for the lysosomal degradation of glycosaminoglycans (GAGS). It hydrolyzes the
non-reducing terminal alpha -L-iduronic acid residues in GAGS including dermatan sulfate and heparan sulfate.  Mature mouse IDUA shares 80% aa identity with human IDUA. Mutations in IDUA that result in enzymatic deficiency lead to the autosomal recessive disease mucopolysaccharidosis type I (MPS I) (1). MPS I can be classified as three clinical subtypes; Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome with decreasing severity, respectively. MPS I causes progressive cellular, tissue and organ damage, and several clinical studies using enzyme replacement therapy show positive results (2, 3). Recently, the IDUA gene has been linked to osteoporosis (4, 5).

1. Scott, H.S. et al. (1995) Hum. Mutat. 6:288.
2. Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.
3. Jameson, E. (2016) Cochrane Database Syst. Rev. 4: CD009354.
4. Kodric, K. et al. (2016) Wien Klin Wochenschr. 128:480.
5. Niu, T. et al. (2016) J. Bone Miner. Res. 31:358.

Publications for alpha-L-Iduronidase/IDUA (9348-GH)(1)

Publication using 9348-GH

• W Acosta, CL Cramer Targeting Macromolecules to CNS and Other Hard-to-Treat Organs Using Lectin-Mediated Delivery Int J Mol Sci, 2020-02-01;21(3):. 2020-02-01 [PMID: 32024082] (In Vivo, Mouse)

Bioinformatics

• Uniprot:
  • P48441()