GALNT7 Protein, Human (HEK293, His)

The GALNT7 protein serves as an indispensable glycopeptidyl transferase for O-linked oligosaccharide biosynthesis. Its enzymatic activity involves catalyzing the transfer of N-acetyl-D-galactosamine residues to pre-existing glycosylated peptides. GALNT7 Protein, Human (HEK293, His) is the recombinant human-derived GALNT7 protein, expressed by HEK293 , with C-6*His labeled tag.

GALNT7 is a glycopeptide transferase that plays a crucial role in O-linked oligosaccharide biosynthesis. Unlike other members of its protein family, GALNT7 does not act as a peptide transferase that directly transfers N-acetyl-D-galactosamine (GalNAc) onto a serine or threonine residue on the protein receptor. Instead, it catalyzes the transfer of GalNAc to an already glycosylated peptide, requiring the prior addition of a GalNAc on a peptide before adding additional GalNAc moieties. While GALNT7 primarily functions in O-linked glycosylation, some peptide transferase activity cannot be entirely excluded, and the identification of its appropriate peptide substrate remains a subject of ongoing investigation.

Measured by its ability to transfer galactose from UDP-GalNAc (HY-114365) to peptide MUC5AC-3/13 that incubate at 37℃ for 20 min. The specific activity is 1288.83 pmol/min/μg.

Materials
Assay buffer: 25 mM Tris, 5 mM MnCl₂, 2.5 mM CaCl₂, pH 7.5
GALNT7 Protein, Human (HEK293, His) (HY-P70867)
Substrates: UDP-GalNAc disodium (HY-114365), MUC5AC-3/13 Peptide
Glycosyltransferase Activity Kit

Procedure
1. Add 40 μL of 1 mM phosphate standard to 360 μL of assay buffer, and dilute the 1 mM phosphate standard provided in the glycosyltransferase kit to obtain a 100 μM stock solution.
2. Prepare a standard curve by performing six consecutive 1:2 dilutions of the 100 μM phosphate stock (diluted with assay buffer). The standard curve ranges from 0.078 to 5 nmol per well.
3. Prepare a reaction mixture consisting of 1 mM UDP-GalNAc, 0.4 mM MUC5AC-3/13 Peptide, and 8 ng/μL coupled phosphatase I in the assay buffer.
4. Dilute GALNT7 to 4 ng/μL with assay buffer.
5. Add 50 μL of different concentration dilutions of the standard curve to a 96-well plate, and add 50 μL of assay buffer to the blank group.
6. Add 25 μL of 4 ng/μL GALNT7 to the 96-well plate; add 25 μL of detection buffer to the blank group.
7. Add 25 μL of the reaction mixture to the wells, excluding the standard curve wells and curve blank wells.
8. Incubate at 37 ℃ for 20 minutes.
9. Add 30 μL of Malachite Green Reagent A to all wells and mix.
10. Add 100 μL of deionized water to all wells and mix.
11. Add 30 μL of Malachite Green Reagent B to all wells, mix, and incubate at room temperature for 20 minutes.
12. Read the plate at 620 nm (absorbance) in endpoint mode.
13. Calculate the specific activity:

Human

HEK293

C-6*His

Q86SF2 (P30-V657)

51809  [NCBI]

Lumenal Domain

PRPDDPSPLSRMREDRDVNDPMPNRGGNGLAPGEDRFKPVVPWPHVEGVEVDLESIRRINKAKNEQEHHAGGDSQKDIMQRQYLTFKPQTFTYHDPVLRPGILGNFEPKEPEPPGVVGGPGEKAKPLVLGPEFKQAIQASIKEFGFNMVASDMISLDRSVNDLRQEECKYWHYDENLLTSSVVIVFHNEGWSTLMRTVHSVIKRTPRKYLAEIVLIDDFSNKEHLKEKLDEYIKLWNGLVKVFRNERREGLIQARSIGAQKAKLGQVLIYLDAHCEVAVNWYAPLVAPISKDRTICTVPLIDVINGNTYEIIPQGGGDEDGYARGAWDWSMLWKRVPLTPQEKRLRKTKTEPYRSPAMAGGLFAIEREFFFELGLYDPGLQIWGGENFEISYKIWQCGGKLLFVPCSRVGHIYRLEGWQGNPPPIYVGSSPTLKNYVRVVEVWWDEYKDYFYASRPESQALPYGDISELKKFREDHNCKSFKWFMEEIAYDITSHYPLPPKNVDWGEIRGFETAYCIDSMGKTNGGFVELGPCHRMGGNQLFRINEANQLMQYDQCLTKGADGSKVMITHCNLNEFKEWQYFKNLHRFTHIPSGKCLDRSEVLHQVFISNCDSSKTTQKWEMNNIHSV

65-80 kDa

Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 50 mM Tris-HCl, 10 mM reduced Glutathione, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.