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Enzymes for O-glycans

OpeRATOR®

OpeRATOR is an O-protease digesting O-glycosylated proteins, with mucin-type O-glycosylation, 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 active on O-glycan proteins with or without sialic acids, although 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 (TNFαR) 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.

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

CASSS MS 2019

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

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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

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.
No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.
The relative abundance and site occupancy of O-glycosylation may vary a lot in a glycoprotein sample, and this will result in the generation of peptides with variable sizes. In some cases, the peptides contain more than one O-glycan, i.e. OpeRATOR does not digest at every site in every molecule. However, the missed digestion sites differ from molecule to molecule, and the compiled data will provide valuable information on the O-glycosylated sites.
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.
Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.

 

Find all FAQ here

Legal and Disclaimers

All rights reserved. Genovis products are covered by one or more patents, pending patent applications, trademarks and/or copyrights owned or controlled by Genovis AB.

For more information about commercial rights, please contact Genovis team at info@genovis.com.

Genovis products are intended for research use only. They are not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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