background:

In the process of producing O -glycosylated biopharmaceuticals , Tn antigen (GalNAc is linked to serine/threonine in an α-linked manner) may appear due to incomplete processing of the core GalNAc residues. Studies have shown that Tn antigen can participate in the binding of C-type lectin macrophage galectin (MGL), and MGL is expressed in dendritic cells and macrophages. Therefore, the linkage of Tn antigen and MGL may cause immunosuppression and help tumors achieve immune escape.

α-N-acetylgalactosidase is a highly specific exoglycosidase that can effectively hydrolyze α-N-acetylgalactosamine (GalNAc) linked to serine or threonine residues in glycoproteins (Tn antigens). The enzyme also shows activity against α1-3 linked terminal GalNAcs. Therefore, α-N-acetylgalactosidase can be used to remove Tn antigens.

α-N-acetylgalactosidase is widely distributed in organisms and participates in the metabolism of glycoconjugates . α-NAGA hydrolyzes GalNAc on glycoproteins under natural conditions. The applicable pH range is 6.0 to 7.6, and no auxiliary factors or special buffer systems are required.

Product Packaging:

Rhinogen ^® α-N-acetylgalactosidase packaging specifications are as follows:

Supporting reagents:

Rhinogen ^® α-N-acetylgalactosidase provides the following reagents:

Product Source:

Recombinantly expressed in E. coli, with a theoretical molecular weight of about 52KD and a 6×His tag at the C-terminus .

Product quality:

SDS-PAGE analysis showed that the purity was ≥95%; no contaminating exoglycosidase, endoglycosidase and protease activities were detected.

Product Features:

α-N-Acetylgalactosaminidas ( exo - α -N- Acetylgalactosaminidase , α-NAGA, EC3.2.1.49) is a highly specific exoglycosidase that effectively hydrolyzes α-N-acetylgalactosamine (GalNAc) linked to serine or threonine residues in glycoproteins (Tn antigen). The enzyme also shows activity against α 1-3 linked terminal GalNAc. α-NAGA hydrolyzes GalNAc on glycoproteins under native conditions. The applicable pH range is 6.0 to 7.6, and no cofactors or special buffer systems are required.

Enzyme activity definition :

α-N-acetylgalactosidase can cleave α-N-acetylgalactosamine from 4-Nitrophenyl N-acetyl-alpha -D- galactosaminide . The enzyme activity is defined by measuring the amount of PNP released at 402 nm. Under the conditions described (20 mM Tris, pH 6.8), each activity unit can release ≥ 600 pmol PNP per minute.

Storage conditions:

1. Transport with ice packs. For long-term storage, please place at -20℃. After resuspending, it can be stored at 2-8℃ for 1 month.

2. Avoid repeated freezing and thawing and frequent temperature changes.

application:

1. Release intact Tn antigen from glycopeptides and glycoproteins;

2. Characterization of Tn antigens for O -glycosylated biopharmaceuticals ;

3. Characterize whether the protein is glycosylated or deglycosylated ;

4. Glycoprotein sequencing and analysis;

5. Recombinant expression of glycoprotein.

characteristic:

α-N-acetylgalactosidase is active in physiological buffers and neutral pH, making it compatible with most samples. In addition, the activity of α-N-acetylgalactosidase is not dependent on any cofactors, such as BSA, which may impair LC-MS analysis. Depending on the nature of the glycoprotein substrate, the incubation time is 2-18h.

Experimental preparation:

The glycoprotein substrate was adjusted to 0.5-5.0 mg/ml in the reaction buffer.

Glycoprotein Digestion:

Take 2000U/unit product as an example:

1. Use 100 μl ddH2O to resuspend the lyophilized powder to 20 U/μl;

2. Add α-N-acetylgalactosidase at a ratio of 1U α-N-acetylgalactosidase/1μg glycoprotein;

3. Incubate at 37°C for 2-18 hours.

Note: Optimize enzyme concentration and incubation time appropriately depending on substrate type.

Note:

1. Avoid repeated freezing and thawing.

2. Appropriate subpackaging can be performed to reduce the loss of activity caused by multiple freeze-thaw cycles.

3. This product is for research use only and is not intended for human or animal diagnosis or treatment.

Frequently asked questions