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OpeRATOR®

OpeRATOR is an O-protease digesting O-glycosylated proteins, with mucin-type O-glycosylation (core 1), N-terminally of the S/T glycosylation site. The presence of O-glycans is required for OpeRATOR to exert its enzymatic activity.

Supplied as a lyophilized powder with no preservatives added, and is available in 2000 unit vials for digestion of up to 2 mg O-glycosylated protein. The enzyme is shipped with 2000 units of SialEXO for removal of sialic acids.

 

 

Detailed Information

OpeRATOR is an endoprotease that catalyzes the hydrolysis of peptide bonds in native mucin-type O-glycosylated proteins in direct proximity of the O-glycans. With high specificity, the endoprotease digests proteins at the N-terminus of O-glycans at serine or threonine. O-glycans of mucin-type are required for OpeRATOR activity and the enzyme will not digest glycoproteins with only N-linked glycans. The enzyme is most active towards sites with asialylated core 1 O-glycans, and it is active on O-glycan proteins with or without sialic acids. However, the activity is higher on O-glycan proteins where sialic acids have been removed. OpeRATOR can be used together with SialEXO (sialidase), which is included in the box.

OpeRATOR is derived from Akkermansia muciniphila and expressed in E. coli. The enzyme contains a His-tag and the molecular weight is 42 kDa. SialEXO is derived from Akkermansia muciniphila and expressed in E. coli. The enzymes in the SialEXO also contain a His-tag and the molecular weights of these components are 43 kDa and 66 kDa, respectively.

Unit Definition

One unit digests ≥ 90% of 1 µg glycoprotein (TNFR) when incubated together with one unit of SialEXO in 20 mM Tris pH 6.8 at 37 °C for 2 h.

Content and Storage

The OpeRATOR box includes:

  • 1 vial of OpeRATOR enzyme (G1-OP1-020) supplied lyophilized in TBS pH 7.6, with no preservatives added.
  • 1 vial of SialEXO (G1-SM1-020) supplied lyophilized in TBS pH 7.6, with no preservatives added.

The box is shipped at ambient temperature, and should be stored at -20°C upon arrival. After reconstitution, the enzymes are stable for 1 month at +4-8 °C.

SmartStories

OpeRATOR changes the game in the field of O-linked Glycoproteomics

Key Characteristics

  • Digests native mucin-type O-glycosylated proteins
  • 2h to overnight (16-18h) reaction
  • Desialylation using SialEXO® (included) increases performance
  • N-terminally of O-glycosylated serine and threonine residues

Applications

Find all applications here

Application Note

OpeRATOR applications download

Download OpeRATOR Application Note

"Determination of O-glycan sites by OpeRATOR digestion and LC-MS"

Download OpeRATOR Posters, Brochures and External References

Antibody Characterization

Poster from CASSS 2019

"In-depth LC-MS Analysis of O-glycosylated Biopharmaceuticals Using an O-glycan Specific Endoprotease"

Download

Brochures and Posters

Download Genovis brochures and posters with the latest details on the SmartEnzymes
 

OpeRATOR references

References

Find external articles and papers using OpeRATOR

 

 

The OpeRATOR Movie

How to Order

For details on ordering please visit How to order. Click add to cart above to buy online.

Quotations

Please do not hesitate to contact us if you need a quotation or have inquiries on price of bulk quantities or licensing opportunities.

Documents

Instructions

Product Specification

Safety Data Sheet

 

 

Certificate of Analysis

FAQ and Support

Popular FAQ

OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl2.

Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

If digestion is insufficient, it could be caused by the O-glycans having a sterically inaccessible location on the sample that the enzyme cannot reach. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, rebuffering, and then digestion with SialEXO and OpeRATOR.

The activity of OpeRATOR is significantly decreased in the presence of sialic acids. We recommend using SialEXO to remove the sialic acids. If this is not an option, then we suggest trying to digest both with and without SialEXO to evaluate the impact for your specific samples and requirements.

 

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