Recombinant Human Fucosyltransferase 7/FUT7 Protein, CF Summary
FUT7 has been formulated so that it can be used in the cell surface glycoengineering of living cells, and does not affect cell viability or native phenotype apart from the intended impact on cell glycobiology.
Details of Functionality
Measured by its ability to transfer fucose from GDP-fucose to fetal bovine fetuin. The specific activity is >175 pmol/min/μg, as measured under the described conditions.
Chinese Hamster Ovary cell line, CHO-derived human Fucosyltransferase 7/FUT7 protein Ala36-Ala342 with and N-terminal human CD33 signal sequence and a C-terminal 6‑His tag
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
<1.0 EU per 1 μg of the protein by the LAL method.
36 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.
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute Coupling Phosphatase 1 to 0.1 mg/mL in Assay Buffer.
Prepare reaction mixture by combining 30 µL of 1.6 mM GDP-Fucose, 120 µL of 50 mg/mL Fetuin, 24 µL of 0.1 mg/mL Coupling Phosphatase 1 and 246 µL of Assay Buffer. This volume is sufficient to assay 10 wells.
Dilute rhFUT7 to 5 µg/mL in Assay Buffer.
Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 μM stock.
Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
Load 15 µL of the 5 µg/mL rhFUT7 into the plate. Include a Control containing 15 µL of Assay Buffer.
Add 35 µL of reaction mixture (step 2) to the wells, excluding the standard curve and curve blank.
Cover the plate with a plate sealer and incubate at 37 °C for 30 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
Add 100 µL of deionized water to all wells. Mix briefly.
Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
Read plate at 620 nm (absorbance) in endpoint mode. 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.
rhFUT7: 0.075 µg
Coupling Phosphatase 1: 0.2 µg
Fetuin: 500 µg
GDP-Fucose: 4000 pmol
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Fucosyltransferase 7/FUT7 Protein, CF
Lewis epitopes are key elements involved in the leukocyte homing and extravasation process and thus are important for lymphocyte maturation and natural defense functions. Fucose-containing glycans also play critical roles in cell signaling and development (1). More than 10 fucosyltransferases have been cloned (2). FUT1 and FUT2 are alpha 1-2 fucosyltransferases and are responsible for ABO blood-group antigen synthesis. FUT8 is an alpha 1-6 fucosyltransferase that adds a fucose to the chitobiose core of N-glycans (3). FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9 are alpha 1-3 or alpha 1-4 fucosyltransferases and are responsible for Lewis antigen generation.
FUT7 plays an exclusive role for the biosynthesis of sialy Lewis X (sLeX) epitope (NeuAc alpha 2,3Gal beta 1,4 [Fuc alpha 1,3] GlcNAc) that serves as a ligand in the E-selectin and P-selectin mediated adhesion of leukocytes to activated endothelium or platelets, and it is critical for the extravasation of immune cells (4, 5, 6). The activity of this enzyme has been measured with a phosphatase-coupled method (7).
R&D Systems Recombinant Human FUT7 has been used for the cell surface glycoengineering of several cell types. In both B cells and mesenchymal stem cells, FUT7 generated, cell surface sLeX leads to enhanced engagement with E-Selectin ligands (8, 9). In naïve regulatory T cells (Treg), engineered sLeX promotes homing to areas of inflammation in vivo (10). These studies suggest that FUT7-mediated generation of sLeX has potential to increase the efficacy of cellular-based therapeutics by enhanced targeting of cells to areas of pathology.
Jafar-Nejad, H. et al. (2010) Glycobiology 20:931.
Becker, D.J. et al. (2003) Glycobiology 13:41R.
Lee, S.H. et al. (2006) J. Biochem. 139:391.
Blander, J. M. et al. (1999) J. Immunol. 163:3746.
Natsuka, S. et al. (1994) J. Biol. Chem. 269:16789.
Sasaki, K. et al. (1994) J. Biol. Chem. 269:14730.
Wu, Z.L. et al. (2011) Glycobiology 21:727.
Silva, M. et al. (2017) J. Immunol. 198:3576.
Pachón-Peña, G. et al. (2017) Stem Cells 35:1080.
Donnelly, C. et al. (2018) Sci. Rep. 420:doi:10.1038/s41598-017-17981-z.
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