GLA/alpha-Galactosidase A Protein, Human (HEK293, His)

GLA/alpha-Galactosidase A Protein, Human (HEK293, His), a recombinant human alpha-Galactosidase A produced in HEK293 cells, has a His tag at the C-terminus. alpha-Galactosidase A is the lysosomal exoglycosidase responsible for the hydrolysis of terminal α-galactosyl residues from glycoconjugates and is the defective enzyme causing Fabry disease^[1].

alpha-Galactosidase A (α-GAL, also known as α-GAL A) is responsible for the breakdown of α-galactosides in the lysosome. Defects in human alpha-Galactosidase A lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of α-galactosylated substrates in the tissues. alpha-Galactosidase A is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy and pharmacological chaperone therapy^[2].

1.Measured by its ability to hydrolyze 4-methylumbelliferyl-alpha -D-galactopyranoside. The specific activity is 8893.70 pmol/min/μg, as measured under the described conditions.
2.Measured by its ability to hydrolyze PNPG. The specific activity is 4275 pmol/min/ug.

Materials
Assay buffer: 50 mM Sodium Citrate, 50 mM NaCl, pH 4.0
Alpha-Galactosidase A Protein, Human (HEK293, His) (HY-P7492)
Substrate: 4-Methylumbelliferyl-α-D-galactopyranoside
Standard: 4-Methylumbelliferone

Procedure
1. Dilute 4-Methylumbelliferone (standard) in assay buffer to concentrations of 0, 312.5, 625, 1250, 2500, 5000 pmol.
2. Add 100 μL of each concentration to the wells.
3. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 365 nm and 445 nm (top read) .
4. Plot the concentration of the standard on the x-axis and the measured RFU on the y-axis to create the standard curve and obtain the standard curve equation.
5. Dilute recombinant Human GLA protein to 0.8 μg/mL in assay buffer.
6. Dilute 4-Methylumbelliferyl-α-D-galactopyranoside to 6.7 mM in assay buffer.
7. Add 50 μL of 0.8 μg/mL Human GLA to the wells and initiate the reaction by adding 50 μL of 6.7 mM substrate. Include a substrate blank with 50 μL of assay buffer and 50 μL of substrate.
8. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 365 nm and 445 nm.
9. Calculate specific activity:

Human

HEK293

C-6*His

P06280 (L32-L429)

2717  [NCBI]

Full Length of Mature Protein

LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL

Approximately 50-60 kDa, based on SDS-PAGE under reducing conditions.

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Solution

Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 150 mM NaCl, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. Fitzmaurice TF, et al. Human alpha-galactosidase A: high plasma activity expressed by the -30G-->A allele. J Inherit Metab Dis. 1997 Sep;20(5):643-57.  [Content Brief]

  [2]. Guce AI, et al. Catalytic mechanism of human alpha-galactosidase. J Biol Chem. 2010 Feb 5;285(6):3625-32.  [Content Brief]