Recombinant R.gnavus alpha-L-Fucosidase His-tag Protein, CF
Recombinant R.gnavus alpha-L-Fucosidase His-tag Protein, CF Summary
Product Specifications
Glu35-Gly583 with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
11385-GH
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
11385-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.
|
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
- Dilute rRg alpha -L-fucosidase to 5 µg/mL with Assay Buffer.
- Dilute Cy5-Lewis X to 0.02 uM with Assay Buffer.
- 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.
- Incubate reaction and control at 37 °C for 30 minutes.
- Add gel loading dye to each tube.
- 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.
- Acquire gel image and determine percent cleavage.
- rRg alpha -L-fucosidase: 0.05 µg
- Cy5-Lewis X: 0.2 pmol
Scientific Data
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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.
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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.
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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.
Background: alpha-L-Fucosidase
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).
- Wu, H. et al. (2021) Cell. Mol. Life Sci. 78:675.
- Sagheddu, V. et al. (2016) Front. Pediatr. 4:57.
- Qin, J. et al. (2010) Nature 464:59.
- Hall, A.B. et al. (2017) Genome Med. 9:103.
- Wu, Z.L. et al. (2020) Glycobiology 30:970.
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