Recombinant E. faecalis O-Glycosidase Protein, CF

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

Recombinant E. faecalis O-Glycosidase Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze p-Nitrophenyl galacto-N-bioside. The specific activity is >12,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived e. faecalis O-Glycosidase protein
  Glu29-Lys1324, with N-terminal Met and 6-His tag
• Accession #: Q833X5
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >75%, 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: 145 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: 128 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 Tris and NaCl.
• Purity: >75%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 0.1 M MES, pH 6.0
  • Recombinant E. faecalis O-Glycosidase (Catalog # 8886-GH)
  • Substrate: p-Nitrophenyl galacto-N-bioside (Sigma, Catalog # N3016), 2 mM stock in deionized water
  • NaOH, 2 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rE. faecalis O-Glycosidase to 1 µg/mL in Assay buffer.
  2. Dilute Substrate to 0.2 mM in Assay buffer.
  3. Load 50 µL of 1 µg/mL rE. faecalis O-Glycosidase in plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
  4. Incubate sealed plate at room temperature for 5 minutes.
  5. Prepare 0.5 M NaOH in deionized water.
  6. Add 100 µL of 0.5 M NaOH to each well to stop the reactions and develop the color.
  7. Read at 405 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x well volume (L) x 10^12 pmol/mol) / (Inc. time (min) x epsilon ** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  *Adjusted for Substrate Blank.
  **Using the extinction coefficient 18100 M^-1cm^-1.
  ***Using the path correction 0.6 cm (based on a 0.0002 L volume).

  Per Reaction:
  • rE. faecalis O-Glycosidase: 0.05 µg
  • Substrate: 0.1 mM

Notes

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

Alternate Names for Recombinant E. faecalis O-Glycosidase Protein, CF

• Endo-alpha-N-Acetylgalactosaminidase
• OGlycosidase
• O-Glycosidase

Background

Enterococcus faecalis O-Glycosidase, also known as endo-alpha -N-Acetylgalactosaminidase, removes O-glycans from glycoproteins. It is broadly active on Core-1, Core-2, Core-3 and Gal-Core-2 structures, which releases the following oligosaccharides, Gal beta 1-3GalNAc, Gal beta 1-3[GlcNAc beta 1-6]GalNAc, GlcNAc beta 1-3GalNAc, Gal beta 1-3[Gal beta 1-3GlcNAc beta 1-6]GalNAc, respectively (1). The enzyme is most active on Core 1, followed by Core 3, then Core 2 structures. The enzyme also has transglycosylation activity and can transfers Core-1 and Core-2 glycans to 1-alkanols, generating alkyl-oligosaccharides. Because the O-Glycosidase is not active on sialylated O-glycans, it is necessary to treat glycoproteins concomitantly with a neuraminidase for deglycosylation purpose (2).

1. Goda, H. M. et al. (2008) Biochem Biophys Res Commun 375:441.
2. Koutsioulis, D. et al. (2008). Glycobiology 18:799.

Publications for O-Glycosidase (8886-GH)(1)

Publication using 8886-GH

• ZL Wu, JM Ertelt Fluorescent glycan fingerprinting of SARS2 spike proteins Scientific Reports, 2021-10-14;11(1):20428. 2021-10-14 [PMID: 34650101] (Bioassay, Human)

Bioinformatics

• Uniprot:
  • Q833X5()