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Recombinant H. pylori Fucosyltransferase Protein, CF

Catalog #: 11072-GT
1 Citation Datasheet
Catalog # Availability Size / Price Qty
11072-GT-020
Recombinant H. pylori Fucosyltransferase Protein Enzyme Activity Diagram.
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Product Details
Citations (1)
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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant H. pylori Fucosyltransferase Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to transfer fucose from GDP-fucose to N-Acetyllactosamine The specific activity is >1500 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived h. pylori Fucosyltransferase protein
Met1-Tyr392, with a C-terminal 6-His tag
Accession #
AAD07710.1
N-terminal Sequence
Analysis
Met1
Predicted Molecular Mass
46 kDa
SDS-PAGE
39 kDa, under reducing conditions.

Product Datasheets

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11072-GT

Product Datasheet

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

11072-GT

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Assay Buffer: 25 mM Tris, 10 mM CaCl2, pH 7.0
  • Recombinant H. pylori Fucosyltransferase (rHp FUT) (Catalog # 11072-GT)
  • Lactosamine (Dextra Laboratories, Catalog # GN204), 50 mM stock in deionized water
  • GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 μL of the 1 mM Phosphate Standard to 360 μL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.
  2. Complete the standard curve by performing six one-half serial dilutions of the 100 μM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare Reaction Mixture containing 2.4 mM Lactosamine, 0.240 mM GDP-Fucose, and 4 μg/mL Coupling Phosphatase 1 (provided in kit) in Assay Buffer.
  4. Dilute rHp FUT to 1 μg/mL in Assay Buffer.
  5. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 μL of 1 μg/mL of rHp FUT into the plate. Include a Control containing 25 μL of Assay Buffer.
  7. Start the reactions by adding 25 μL of Reaction Mixture to the wells, excluding the standard curve.
  8. Incubate sealed plate at 37 °C for 20 minutes.
  9. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 μL of deionized water to all wells.
  11. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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.

Per Reaction:

  • rHp FUT: 0.025 µg
  • Coupling Phosphatase 1: 0.1 μg
  • Lactosamine: 1.2 mM
  • GDP-Fucose: 0.12 mM

Scientific Data

Enzyme Activity View Larger

Recombinant H. pylori Fucosyltransferase His-tag Protein (Catalog # 11072-GT) Enzyme Activity Diagram.

Enzyme Activity View Larger

Asialofetuin (4 μg) was incubated with 0.2 nmol of GDP-Cy3-Fucose (ES401) with or without rHp. FUT (Catalog # 11072) (1.0 μg) in 25 μL of 25 mM Tris pH 7.5, 10 mM MnCl2 at 37 °C for 30 minutes. Following labeling, half of the AF was deglycosylated with PNGase F N-glycan Releasing Kit (EA006) and resolved in 15% SDS-PAGE. Left side is the TCE image and right side is the Fluorescent image of the same gel.

SDS-PAGE View Larger

2 μg/lane of Recombinant H. pylori Fucosyltransferase Protein (Catalog # 11072-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 39 kDa.

Background: Fucosyltransferase

Lewis X (LeX), a fucosylated trisaccharide glycan epitope (Gal beta 1,4 [Fuc alpha 1,3]GlcNAc beta ) also known as CD15, and sialylated Lewis X (sLeX) are distributed throughout eukaryotes and are determinants of many functional glycoconjugates that play central roles in numerous physiological and pathological processes (1, 2). Lex-bearing glycans are also found in the infectious bacterium Helicobacter pylori to mask the bacterium from the host immune surveillance (3). H. pylori is the pathogen that causes peptic ulcers that can further lead to stomach cancer and gastritis. H. pylori fucosyltransferase is responsible for generating the LeX glycans, therefore is potentially a drug target for curing peptic ulcers, gastritis and stomach cancer. In molecular terms, H. pylori fucosyltransferase is an  alpha 1,3 fucosyltransferase that shares function with human FUT4, FUT5, FUT6, and FUT9 (4). Remarkably, H. pylori fucosyltransferase can transfer IgG antibody to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose (5). The activity of this enzyme has been measured with a phosphatase coupled method (6).

References
  1. Gooi, H.C. et al. (1981) Nature 292:156.
  2. Phillips, M.L. et al. (1990) Science 250:1130.
  3. Moran, A.P. et al. (1996) FEMS Immunol Med Microbiol 16:105.
  4. de Vries, T. et al. (2001). Glycobiology 11:119R.
  5. Li, J. et al. (2018) ACS Cent. Sci. 4:1633.
  6. Wu, Z.L. et al. (2011) Glycobiology 21:727.
Long Name
alpha-(1,3) Fucosyltransferase FucT
Alternate Names
Fucosyltransferase; FucT

Citation for Recombinant H. pylori Fucosyltransferase Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering
    Authors: Su, T;Chua, WZ;Liu, Y;Fan, J;Tan, SY;Yang, DW;Sham, LT;
    Science advances
    Species: Bacteria
    Sample Types: Whole Cells
    Applications: Enzyme Activity

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