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

2 μg/lane of Recombinant Cynomolgus alpha-L-Iduronidase/IDUA Protein (Catalog # 11281-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at ~81-90 kDa.

• Applications: Enzyme Activity
• Format: Carrier-Free

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

• Additional Information: His-tag
• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl alpha -L-iduronide. The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived cynomolgus monkey alpha-L-Iduronidase/IDUA protein
  Ala26-Pro653 with a C-terminal 10-His tag
• Accession #: XP_005554313.1
• N-terminal Sequence: Ala26 & Glu27
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 72 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: 81-90 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 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 Sodium Acetate, 150 mM NaCl, 0.02% Brij-35 (w/v), pH 3.5
  • Development Buffer: 0.1 M Tris, pH 9.0
  • Recombinant Cynomolgus alpha-L-Iduronidase/IDUA (rcynoIDUA) (Catalog # 11281-GH)
  • Substrate: 4-methylumberlliferyl-alpha -L-Iduronide, 20 mM stock in DMSO
  • Black 96-well Plate
  • Fluorescent Plate Reader
  1. Dilute rcynoIDUA to 0.2 µg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.
  2. Dilute Substrate to 800 µM in Assay Buffer.
  3. Combine equal volumes of 0.2 µg/mL rcynoIDUA and 800 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.
  4. Incubate reactions and Substrate Blank at room temperature for 10 minutes.
  5. Dilute reactions to 0.005 µg/mL rcynoIDUA in Developing Buffer. Dilute the Substrate Blank similarly.
  6. Load 100 µL of the diluted reactions and Substrate Blank into a plate.
  7. Read plate 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 from calibration standard 4-methylumbelliferone.
  Per Well:
  • rcynoIDUA: 0.0005 µg
  • Substrate: 20 µM

Notes

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

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

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

Background

alpha -L-Iduronidase is a member of the glycoside hydrolase family encoded by the IDUA gene (1). It is an important enzyme required for the lysosomal degradation of glycosaminoglycans (GAGS) and hydrolyzes the non-reducing terminal alpha -L-iduronic acid residues in GAGS including dermatan sulfate and heparan sulfate.  Cynomolgus monkey IDUA is a 653 aa protein composed of a signal peptide removed in the lysosome for mature form and three domains: a triosephosphate isomerase barrel fold containing the catalytic site, a beta -sandwich domain, and an Ig(Ig)-like domain. The protein has six reported N-glycosylation sites and the glycosylation status of the enzyme correlates with its catalytic activity (1). More than a hundred disease-associated variants in the IDUA gene have been identified (1, 2). Mutations in IDUA that result in enzymatic deficiency lead to the autosomal recessive disease mucopolysaccharidosis type I (MPS I) (3). MPS I can be classified into 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 (4, 5). More recently, the IDUA gene has been linked to osteoporosis (6, 7). 

1. Maita, N. et al. (2013) Proc. Natl. Acad. Sci. 110:14628.
2. Borges, P et al. (2021) Front. Mol. Biosci. 8:752797.
3. Scott, H.S. et al. (1995) Hum. Mutat. 6:288.
4. Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.
5. Jameson, E. (2016) Cochrane Database Syst. Rev. 4: CD009354.
6. Kodric, K. et al. (2016) Wien Klin Wochenschr. 128:480.
7. Niu, T. et al. (2016) J. Bone Miner. Res. 31:358.

Bioinformatics

• Uniprot:
  • XP_005554313.1()