Recombinant B. Thetaiotaomicron Arylsulfatase Protein, CF

Recombinant B. Thetaiotaomicron Arylsulfatase Protein, CF (Catalog # 11383-SU) has specificity for 3S-Galactose. It has been shown to remove 3’-sulfate (3S) from various glycan structures including LacNAc, LNB, Lewis-X and Lewis-A.

Lane 1 contained fluorescent substrate glycan Cy5-Fuc labeled sulfo-G2. Following treatment with Recombinant B. Thetaiotaomicron Arylsulfatase Protein, CF (Catalog # 11383-SU) 3’-sulfate (3S) from galactose was removed and a mobility shift was observed. SDS-PAGE gel was imaged using the red fluorescent channel.

2 μg/lane of Recombinant Bacteroides Thetaiotaomicron Arylsulfatase His-tag Protein (Catalog # 11383-SU) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 54-60 kDa under reducing condition.

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

Recombinant B. Thetaiotaomicron Arylsulfatase Protein, CF Summary

• Additional Information: His-tag
• Details of Functionality: Measured by its ability to cleave 3'-sulfate from Galactose.
  Recombinant Bt.1636 arylsulfatase will cleave >50% Cy5-Fuc labeled sulfo-G2 (Catalog # GL305), as measured under the described conditions.
• Source: E. coli-derived b. thetaiotaomicron Bacteroides Thetaiotaomicron Arylsulfatase protein
  Met1-Lys509 (S77C) with a C-terminal 6-His tag
• Accession #: WP_011107907.1
• N-terminal Sequence: Met1 & Leu8
• 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: 58 kDa & 59 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: 54-60 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, pH 5.5
  • Recombinant Bt.1636 arylsulfatase (rBt. 1636 arylsulfatase) (Catalog # 11383-SU)
  • Cy5-Fuc labeled sulfo-G2 (Cy5-sulfoG2) (Catalog # GL305)
  • 15% SDS-PAGE gel
  • Gel loading dye
  • Gel Imager with Cy5 fluorescent dye detection capability
  1. Dilute rBt. 1636 arylsulfatase to 0.05 µg/µL with Assay Buffer.
  2. Dilute Cy5-sulfoG2 to 0.02 uM with Assay Buffer.
  3. For reaction, combine 10 µL of rBt. 1636 arylsulfatase and 10 µL of Cy5-sulfoG2. For a control, combine 10 µL of Assay Buffer and 10 µL Cy5-sulfoG2.
  4. Incubate reaction and control at 37 °C for 1 hour.
  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 with a gel imager.
  8. Determine intensities of each band. Calculate percent cleavage of Cy5-sulfoG2:
  
  

  % Cleavage = [ 1 - (	Intensity of uncleaved Cy5-sulfoG2	) ] x 100
  	Intensity of cleaved and uncleaved bands

  Per Reaction:
  • rBt. 1636 arylsulfatase: 0.5 µg
  • Cy5-sulfoG2: 0.2 pmol

Notes

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

Alternate Names for Recombinant B. Thetaiotaomicron Arylsulfatase Protein, CF

• Bacteroides Thetaiotaomicron Arylsulfatase
• BT1636

Background

Recombinant Bacteroides thetaiotaomicron Arylsulfatase His-tag, known in the literature as BT1636, is a cell surface-localized member of a diverse sulfatase family involved in mucin degradation (1). BT1636 is active on 3'S-Gal-beta 1,3-GalNAc (core 1) as well as more complex sulfated glycans built around other core structures (1). This broader specificity suggests the enzyme evolved to recognize various linkages and substitutions found in O-glycans. BT1636 displays an alpha / beta / alpha topology with a C-terminal sub-domain, a coordinated calcium ion, a deep pocket for substrates, and an active site with highly conserved residues that interact with the sulfate group (1-3). Bacteroides thetaiotaomicron (Bt) is one of the major bacteria of the human gut microbiome that can utilize O-glycans as a sole source of nutrients and has a requirement for active sulfatases for competitive colonization (4, 5). Although the Bt genome encodes 28 different S1 sulfatases (1), investigation suggests BT1636 is critical as an early enzyme in O-glycan cleavage that allows import and further degradation of O-glycans in the periplasmic space. Bt mutants lacking only sulfatase BT1636 displayed a large defect in colonization competition experiments versus wild-type Bt (1). Given BT1636 is critical to initiation of the complex pathway of mucin degradation, target and inhibition of this enzyme may lead to the ability to inhibit pathways that enable and contribute to disease such as IBD (1). The activity of recombinant BT1636 is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled glycan as the substrate (6).

1. Luis, A.S. et al. (2021) Nature. 598:332.
2. Cartmell, A. et al. (2017) Proc. Natl. Acad. Sci. U.S.A. 27:7037.
3. Ulmer, J.E. et al. (2014) J. Biol. Chem. 289:24289.
4. Martens, E.C. et al. (2008) Cell Host Microbe 4:447.
5. Benjdia, A. et al. (2011) J. Biol. Chem. 286:25973.
6. Wu, Z.L. et al. (2020) Glycobiology 30:970.

Bioinformatics

• Uniprot:
  • WP_011107907.1()