Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein, CF

Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein (Catalog # 11261-GT) introduces an alpha 1-3 linked galactose residue to terminal lactosamine (LN) structure and generates alpha -Gal epitope (boxed). alpha -Gal is ubiquitously presented in non-primate mammals but absent in humans and is responsible for hyperacute rejection of organs transplanted from alpha -gal donors. The enzyme shows reduced activity on lactose, which is the basis for the current enzyme activity assay.

2 μg/lane of Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein (Catalog # 11261-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 39‑48 kDa.

• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer galactose from UDP-galactose to alpha -Lactose. The specific activity is >400 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived porcine alpha3GalT/GGTA1 protein
  Glu23-Ile371, with a C-terminal 6-His tag
• Accession #: P50127-1
• N-terminal Sequence: Glu23
• 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: 42 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: 39-48 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Phosphate Buffer 1: 10X solution of 250 mM Tris, 100 mM CaCl₂, pH 7.5 (provided in kit)
  • MnCl₂: 100 mM MnCl₂ in deionized water (provided in kit)
  • Recombinant Porcine alpha 3GalT/GGTA1 His-tag (rpGGTA-1) (Catalog # 11261-GT)
  • UDP-Galactose, 10 mM stock in deionized water
  • alpha -Lactose, 0.3 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader
  1. Prepare 1X Assay Buffer (25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂, pH 7.5) containing 10 mM MnCl₂ by combining equal volumes of Phosphate Buffer 1 and 100 mM MnCl₂ and diluting 5-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Dilute Coupling Phosphatase 1 (provided in kit) to 10 µg/mL in 1X Assay Buffer.
  5. Prepare a Reaction Mixture containing 0.8 mM UDP-Galactose, 40 mM alpha -Lactose, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
  6. Dilute rpGGTA-1 to 5 µg/mL in 1X Assay Buffer.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  8. Load 25 µL of 5 µg/mL rpGGTA-1 into empty wells of the same plate as the curve. Include a Control containing 25 µL of 1X Assay Buffer.
  9. Start the reaction by adding 25 µL of Reaction Mixture to all wells, excluding standard curve.
  10. Seal and incubate plate at 37 °C for 20 minutes.
  11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  12. Add 100 µL of deionized water to all wells. Mix briefly.
  13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  14. Read plate at 620 nm (absorbance) in endpoint mode.
  15. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

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

  Per Reaction:
  • rpGGTA-1 0.125 µg
  • Coupling Phosphatase 1: 0.1 µg
  • UDP-Galactose 0.4 mM
  • alpha -Lactose: 20 mM

Notes

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

Alternate Names for Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein, CF

• alpha Gal
• alpha3GalT
• GALT
• GGTA1
• Glt6d1

Background

GGTA1, also designated as alpha 3GalT, is an alpha ­1,3 galactosyltransferase that catalyzes the transfer of galactose from UDP­ alpha ­D­galactose to various glycoconjugates to form non­reducing terminal alpha ­1,3­linked galactosyl moieties, or alpha -Gal epitope (1). This enzyme is expressed in many mammalian species but is absent from humans, apes, and Old World monkeys due to the mutational inactivation of the gene (2). Therefore, humans do not have alpha -Gal on glycoconjugates, but produce large amounts of antibody to this epitope, resulting from exposure to alpha -Gal containing antigens (3). GGTA1 is of great medical interest because the immune responses may be enhanced when selected targets are decorated with alpha -Gal structures (4). Additionally, the presence of anti-­ alpha -­Gal antibodies is a barrier to xenotransplantation of organs. Recombinant pig GGTA1 may provide easy access to a wide spectrum of alpha -Gal epitopes and their derivatives to support studies on xenotransplantation and other pharmaceutical research (5, 6). Structurally and mechanistically GGTA1 is a model for several homologous glycosyltransferases that differ in donor and acceptor substrate specificity, including the histo blood group A and B glycosyltransferases, Forssman glycolipid synthase, and isogloboside 3 synthase (7).

1. Larsen R.D. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:8227. 
2. Joziasse, D.H. et al. (1992) J. Biol. Chem. 267:5534. 
3. Wigglesworth K.M. et al. (2011) J. Immunol. 186:4422. 
4. Dequchi, T. et al. (2010) Cancer Res. 70:5259. 
5. Anraku, K. et al. (2017) Org. Biomol. Chem. 15:2979. 
6. Galili, U. (2021) Front. Mol. Biosci. 8:746883. 
7. Breton, C. et al. (2005) Glycobiology 16:29R.  

Bioinformatics

• Uniprot:
  • P50127-1()