Recombinant Mouse Lysosomal alpha-Glucosidase/GAA, CF

R&D Systems | Catalog # 11400-GH

His-tag
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Key Product Details

  • R&D Systems CHO-derived Recombinant Mouse Lysosomal alpha-Glucosidase/GAA (11400-GH)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

P70699.2

Applications

Enzyme Activity
View all Lysosomal alpha-Glucosidase Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived mouse Lysosomal alpha-Glucosidase protein
Glu70-Ser953 with a N-terminal 6-His tag

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.

N-terminal Sequence Analysis

His

Predicted Molecular Mass

99 kDa

SDS-PAGE

97-107 kDa, under reducing conditions

Activity

Measured by its ability to release glucose from starch.
The specific activity is >5000 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Mouse Lysosomal alpha-Glucosidase/GAA, CF

Recombinant Mouse Lysosomal alpha -Glucosidase His-tag Protein Enzyme Activity Diagram.

Recombinant Mouse Lysosomal alpha -Glucosidase His-tag Protein, CF (Catalog # 11400-GH) hydrolyses both alpha-1,4- and alpha-1,6-glucosidic linkages on glycogen to release terminal glucose.

Recombinant Mouse Lysosomal alpha ‑Glucosidase His-tag Protein SDS-PAGE.

2 μg/lane of Recombinant Mouse Lysosomal alpha ‑Glucosidase His-tag Protein (Catalog # 11400-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 97-107 kDa, under reducing conditions.

Formulation, Preparation, and Storage

11400-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. 
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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: Lysosomal alpha-Glucosidase

Acid alpha-glucosidase (GAA) is an essential enzyme for the hydrolysis of glycogen alpha 1-4 and alpha 1,6-glycosidic linkages within the lysosome (1,2). GAA is a member of the glycoside hydrolase family GH31 and contains an N-terminal trefoil-P domain, a beta -sheet domain, a catalytic barrel, and two C-terminal beta -sheet domains (2). In addition to an active site and substrate binding domain, GAA has an additional reported secondary substrate-binding domain that may enhance the processivity of the enzyme (2). Mouse GAA has approximately 80% homology with human GAA. Defects in GAA cause glycogen storage disease II, also known as Pompe's disease, which is a rare autosomal recessive metabolic disorder that damages muscle and nerve cells due to accumulation of glycogen in the lysosome (3). Pompe disease occurs in babies, children, and adults who inherit a defective GAA gene and affects an estimated 5,000 to 10,000 people worldwide (4). Enzyme replacement therapy (ERT) is used to treat patients with Pompe disease and other lysosomal storage diseases (LSDs) (5, 6). Alternative therapeutic strategies such as pharmacological chaperone therapy (PCT) are being explored for use in concert with or independently for the potential to stabilize the target enzyme without impact to the catalytic activity (2, 7, 8).

References

  1. Hoefsloot, L.H. et. al. (1988) EMBO J. 7:1697.
  2. Roig-Zamboni, V. et. al. (2017) Nat. Commun. 8:1111.
  3. Wan, L. et. al. (2008) J. Neurol. 255:831.
  4. Fukuda, T. et. al. (2007) Curr. Neurol. Neurosci. Rep. 7:71.
  5. Van Gelder, C.M. et. al. (2014) J. Inherit. Metab. Dis. In press.
  6. Toscano, A. and B. Schoser. (2013) J. Neurol. 260:951.
  7. Porto, C. et. al. (2012) Mol. Ther. 20:2201.
  8. Parenti, G. et. al. (2015). Mol. Ther. 23:1138.

Long Name

Glucosidase, Alpha; Acid

Alternate Names

Acid alpha-Glucosidase, Acid Maltase, GAA, LYAG, Lysosomal alphaGlucosidase

Entrez Gene IDs

2548 (Human); 14387 (Mouse); 367562 (Rat); 102141245 (Cynomolgus Monkey)

Gene Symbol

GAA

UniProt

P70699.2

Additional Lysosomal alpha-Glucosidase Products

Product Documents for Recombinant Mouse Lysosomal alpha-Glucosidase/GAA, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Mouse Lysosomal alpha-Glucosidase/GAA, CF

For research use only

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Protocols

View specific protocols for Recombinant Mouse Lysosomal alpha-Glucosidase/GAA, CF (11400-GH):

Materials
  • Assay Buffer: 0.1 M Sodium Acetate, pH 4.5
  • Recombinant Mouse Lysosomal alpha-Glucosidase/GAA (rmGAA) (Catalog # 11400-GH)
  • Substrate: Starch from potato, 2% (w/v) stock in deionized water
  • Stop Solution: 4.4 mM Dinitrosalicylic Acid, 1 M Potassium Tartrate, 0.4 M Sodium Hydroxide in deionized water 
  • Maltose Standard, 20 mM stock in deionized water 
  • 96 well Clear Plate  (Catalog # DY990)
  • Plate Reader with Absorbance Read Capability
  1. Dilute 20 mM Maltose standard by adding 200 µL of 20 mM Maltose Standard to 600 µL of Assay Buffer for a 5 mM stock. This is the first point of the standard curve. 
  2. Prepare the standard curve by performing five one-half serial dilutions of the 5 mM Maltose stock in Assay Buffer. Make sure there are 400 μL in each tube for each point of the curve (remove 400 μL from the last point of the curve). Prepare one tube with only 400 μL of Assay Buffer for the curve blank. The standard curve has a range of 19.5 to 625 nmol per well. 
  3. Dilute rmGAA to 32 μg/mL in Assay Buffer. 
  4. Dilute 2% starch to 1.5% in Assay Buffer.
  5. Prepare reactions by combining 20 μL of diluted rmGAA with 380 μL of 1.5% starch (step 4). Include a control by combining 20 μL of Assay Buffer with 380 μL of 1.5% starch. 
  6. Vortex, spin, and then incubate reactions, control, and standard curve at 37 °C for 1 hour. 
  7. Add 400 μL of Stop Solution to all vials, including standard curve. 
  8. Heat all vials at 95-100 °C for 6 minutes. Then, cool on ice. Tip: Use lid-locks to keep vials closed when heating. 
  9. Load 250 µL of each dilution of the standard curve, reactions, and controls to empty wells in clear plate. 
  10. Read plate at 546 nm (absorbance) in endpoint mode. 
  11. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted glucose produced* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)

    


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

Per Well

  • rmGAA: 0.2 μg
  • Starch: 0.71%
















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