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

Catalog #: 11201-GH Datasheet / COA / SDS

CHO-expressed (aa 28-653 H33Q)

Catalog #	Availability	Size / Price	Qty
11201-GH-020

Recombinant Human alpha-L-Iduronidase/IDUA His-tag Protein SDS-PAGE.

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Product Details

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

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

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl alpha -L-iduronide. The specific activity is >6000 pmol/min/μg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived human alpha-L-Iduronidase/IDUA protein
Ala28-Pro653 (His33Gln), with a C-terminal 10-His tag

Accession #

P35475.2

N-terminal Sequence
Analysis

Ala28

Predicted Molecular Mass

71 kDa

SDS-PAGE

80-91 kDa, under reducing conditions.

Product Datasheets

11201-GH

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

11201-GH

Formulation	Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Assay Procedure

Materials

Assay Buffer: 50 mM NaOAc, 150 mM NaCl, 0.02% Brij-35 (w/v), pH 3.5
Development Buffer: 0.1 M Tris, pH 9.0
Recombinant Human IDUA (rhIDUA) (Catalog # 11201-GH)
Substrate: 4-methylumberlliferyl-alpha -L-Iduronide, 20 mM stock in DMSO
Black 96-well PlateFluorescent Plate Reader

Dilute rhIDUA to 0.2 µg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.
Dilute Substrate to 800 µM in Assay Buffer.
Combine equal volumes of 0.2 µg/mL rhIDUA and 800 µM Substrate. Include a Substrate Blank by combining equal volumes of Assay Buffer and 800 µM Substrate.
Incubate reactions and Substrate Blank at room temperature for 10 minutes.
Dilute rhIDUA/substrate mixtures to 0.005 µg/mL of rhIDUA in Developing Buffer.
Load 100 µL of the diluted rhIDUA/substrate mixtures into a plate.
Read plate at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
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:

rhIDUA: 0.0005 µg
Substrate: 20 µM

Scientific Data

SDS-PAGE

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2 μg/lane of Recombinant Human alpha-L-Iduronidase/IDUA His-tag Protein (Catalog # 11201-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 80-91 kDa.

Reconstitution Calculator

Background: alpha-L-Iduronidase/IDUA

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 a-L-iduronic acid residues in GAGS including dermatan sulfate and heparan sulfate. Human 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 B-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). H33Q, present in the recombinant product, represents a benign and common polymorphism (8).

References