Recombinant R.gnavus alpha-L-Fucosidase His-tag Protein, CF

Recombinant R. gnavus alpha-L-Fucosidase His-tag Protein, CF (Catalog # 11385-GH) has specificity for both Lewis A ( alpha 4) and Lewis X ( alpha 3) link fucose. Sialyation of Lewis A or Lewis X does not affect the substrate recognition of alpha -L-fucosidase.

2 μg/lane of Recombinant R. gnavus alpha ‑L‑Fucosidase His-tag Protein (Catalog # 11385-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 62-68 kDa under reducing conditions.

Lane 1 contained fluorescent substrate glycan Cy5-Fuc labeled Lewis X. Following treatment with Recombinant R. gnavus alpha-L-Fucosidase His-tag Protein, CF (Catalog # 11385-GH) both alpha 3 linked Fucose on GlcNAc were removed and a mobility shift was observed. SDS-PAGE gel was imaged using the red fluorescent channel.

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

Recombinant R.gnavus alpha-L-Fucosidase His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave alpha 3 Fucose.
  Recombinant a-L-Fucosidase will cleave >50% Cy5-Fuc labeled Lewis X (Catalog # GL306), as measured under the described conditions.
• Source: E. coli-derived alpha-L-Fucosidase protein
  Glu35-Gly583 with an N-terminal Met and 6-His tag
• Accession #: WP_243035407.1
• N-terminal Sequence: Met
• 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: 62 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: 62-68 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:
  • Assay Buffer: 50 mM MES, 10 mM CaCl₂, 10 mM MnCl₂, pH 6.0
  • Recombinant R. gnavus alpha -L-fucosidase (rRg alpha -L-fucosidase) (Catalog # 11385-GH)
  • Cy5-Fuc labeled Lewis X (Cy5-Lewis X) (Catalog # GL306)
  • 15% SDS-PAGE
  • Gel loading dye
  • Gel Imager with Cy5 fluorescent dye detection capability
  1. Dilute rRg alpha -L-fucosidase to 5 µg/mL with Assay Buffer.
  2. Dilute Cy5-Lewis X to 0.02 uM with Assay Buffer.
  3. For reaction, combine 10 µL of rRg alpha -L-fucosidase and 10 µL of Cy5-Lewis X. For control, combine 10 µL of Assay Buffer and 10 µL of Cy5-Lewis X.
  4. Incubate reaction and control at 37 °C for 30 minutes.
  5. Add gel loading dye to each tube.
  6. Load half the volume of each reaction and control onto a 15% SDS-PAGE gel. Let samples migrate at least 80% down the gel before stopping.
  7. Acquire gel image and determine percent cleavage.
  Per Reaction:
  • rRg alpha -L-fucosidase: 0.05 µg
  • Cy5-Lewis X: 0.2 pmol

Notes

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

Alternate Names for Recombinant R.gnavus alpha-L-Fucosidase His-tag Protein, CF

• alphaLFucosidase
• alpha-L-Fucosidase

Background

Recombinant R. gnavus alpha -L-Fucosidase His-tag ( alpha -L-Fuc) catalyzes the hydrolysis of terminal alpha -L-Fucose. alpha -L-Fuc is monomeric and consists of two distinct domains, an N-terminal catalytic domain comprising residues 46-366 and a C-terminal F5/8 Type C domain covering residues 385-526 (1). Residues 23-45 wrap around the C-terminal beta -sandwich domain (1). R. gnavus is an early colonizer of the human gut but persists in healthy adults (2-3). An increasing number of studies are reporting a disproportionate representation of R. gnavus in diseases, such as inflammatory bowel disease (4). To access a source of nutrients, gut bacteria encode alpha ‑L‑fucosidases that catalyze the hydrolysis of terminal alpha -L-fucosidic linkages. alpha -L-Fuc has the capacity to recognize fucosylated glycans and to hydrolyze both alpha 1‑3 (Lewis X) and alpha 1–4 (Lewis A) fucosyl linkages although the preferred substrate is sialyated Lewis X epitope (1). This fucosidase specificity can potentially be exploited for use in human disease diagnostic assays, as a tool to identify N-glycan biomarkers of disease, and for glycoprofiling biopharmaceutical glycoproteins. The activity of alpha -L-Fuc is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled glycan Cy5-Fuc labeled Lewis X as the substrate (5).

1. Wu, H. et al. (2021) Cell. Mol. Life Sci. 78:675.
2. Sagheddu, V. et al. (2016) Front. Pediatr. 4:57.
3. Qin, J. et al. (2010) Nature 464:59.
4. Hall, A.B. et al. (2017) Genome Med. 9:103.
5. Wu, Z.L. et al. (2020) Glycobiology 30:970.

Bioinformatics

• Uniprot:
  • WP_243035407.1()