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

Recombinant Human alpha-L-Iduronidase/IDUA Protein, CF (Catalog # 4119-GH) hydrolyzes iduronic acid from dermatan and heparan sulfates.

Recombinant Human alpha-L-Iduronidase/IDUA Protein, CF (Catalog # 4119-GH) is measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl alpha -L-iduronide.

2 μg/lane of Recombinant Human alpha-L-Iduronidase / IDUA Protein (4119-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at ~83 kDa under reducing conditions.

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

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

• Additional Information: NS0-expressed
• 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/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human alpha-L-Iduronidase/IDUA protein
  Ala26-Pro653 (Ala26Thr), with a C-terminal 10-His tag
• Accession #: AAA81589
• N-terminal Sequence: Thr26
• Protein/Peptide Type: Recombinant Enzymes
• Gene: IDUA
• 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: 71 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 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 Human alpha ‑L‑Iduronidase/IDUA (rhIDUA) (Catalog # 4119-GH)
  • Substrate: 4-methylumberlliferyl-alpha -L-iduronide (Glycosynth, Catalog # 44076), 20 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhIDUA 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 rhIDUA 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 in Developing Buffer.
  6. Load 100 µL of the diluted mixtures into a plate.
  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:
  • rhIDUA: 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 Human 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. Mutations in IDUA that result in enzymatic deficiency lead to the autosomal recessive disease mucopolysaccharidosis type I (MPS I) (1). MPS I causes progressive cellular, tissue and organ damage, and several clinical studies using enzyme replacement therapy have shown promising benefits (2).

1. Scott, H.S. et al. (1995) Hum. Mutat. 6:288.
2. Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.

Publications for alpha-L-Iduronidase/IDUA (4119-GH)(3)

Publications using 4119-GH

• K Laoharawee, KM Podetz-Ped, TT Nguyen, SM Singh, MC Smith, LR Belur, WC Low, KF Kozarsky, RS McIvor Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome Molecular genetics and metabolism reports, 2023-01-20;34(0):100956. 2023-01-20 [PMID: 36704405] (Bioassay, Mouse)
• Y Osaki, A Saito, S Kanemoto, M Kaneko, K Matsuhisa, R Asada, T Masaki, K Orii, T Fukao, S Tomatsu, K Imaizumi Shutdown of ER-associated degradation pathway rescues functions of mutant iduronate 2-sulfatase linked to mucopolysaccharidosis type II Cell Death Dis, 2018-07-24;9(8):808. 2018-07-24 [PMID: 30042467] (Enzyme Assay, Human)
• K Laoharawee, RC DeKelver, KM Podetz-Ped, M Rohde, S Sproul, HO Nguyen, T Nguyen, SJ St Martin, L Ou, S Tom, R Radeke, KE Meyer, MC Holmes, CB Whitley, T Wechsler, RS McIvor Dose-Dependent Prevention of Metabolic and Neurologic Disease in Murine MPS II by ZFN-Mediated In�Vivo Genome Editing Mol. Ther., 2018-03-10;26(4):1127-1136. 2018-03-10 [PMID: 29580682] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: IDUA
• Uniprot:
  • AAA81589()