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rS. equi subsp. zooepidemicus IgG endopeptidase IdeZ, CF

Catalog #: 11572-IZ Datasheet / COA / SDS
Recombinant Streptococcus His-tag
Catalog # Availability Size / Price Qty
11572-IZ-050

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Recombinant Streptococcus equi subsp. zooepidemicus IgG endopeptidase IdeZ His-tag Protein SDS-PAGE.
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rS. equi subsp. zooepidemicus IgG endopeptidase IdeZ, CF Summary

  • R&D Systems E. coli-derived rS. equi subsp. zooepidemicus IgG endopeptidase IdeZ (11572-IZ)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<0.10 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave Human IgG.
The DC50 is <25 ng, as measured under the described conditions. The DC50 is defined as the amount of enzyme required to cleave 50% of 1 μg human IgG in 30 minutes at 37 °C. Use of Recombinant Streptococcus equi subsp. zooepidemicus IdeZ in the cleavage of other IgGs may required alternative conditions for optimal performance.
Source
E. coli-derived IdeZ protein
Asp35-Ser349, with an N-terminal Met and C-terminal 6-His tag
Accession #
WP_014622780.1
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
36 kDa
SDS-PAGE
35-39 kDa, under reducing conditions

Product Datasheets

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11572-IZ

Product Datasheet
COA
SDS

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.

11572-IZ

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with dry ice or equivalent. 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
  • Assay Buffer: 25 mM Sodium Phosphate, 150 mM NaCl, pH 7.5
  • Recombinant S. equi subsp. zooepidemicus IgG endopeptidase IdeZ (rS.e IdeZ) (Catalog # 11572-IZ)
  • Purified Human IgG (Catalog # 1-001-A)
  • 15% SDS-PAGE gel
  • Reducing Sample Buffer
  • Gel Staining Reagent
  1. Prepare a curve of rS.e IdeZ by diluting rS.e IdeZ to 200, 50, 12.5, 3.125, 0.781, 0.195, and 0.049 µg/mL in Assay Buffer.
  2. Dilute Human IgG to 200 µg/mL in Assay Buffer.
  3. Combine 20 µL of each rS.e IdeZ curve dilution and 20 µL of 200 µg/mL Human IgG. Include a control by combining 20 µL of 200 µg/mL Human IgG and 20 µL Assay Buffer.
  4. Incubate reaction mixtures and control at 37 °C for 30 minutes.
  5. Combine each 10 µL reaction mixture (including controls) with 10 µL of Reducing Sample Buffer.  Heat at 95 °C for 3 minutes.
  6. Load entire volume of each reaction mixture (20 µL) per lane onto a 15% SDS-PAGE gel and perform electrophoresis.
  7. Stain gel and analyze the percent digestion of Human IgG using densitometry for each rS.e IdeZ curve dilution.
  8. Determine the DC50 by plotting % digestion vs rS.e IdeZ concentration (ng) using 4-PL fitting.
Per Lane:
  • rS.e IdeZ: 1000, 250, 62.5, 15.6, 3.9, 0.98, 0.24, and 0 ng
  • Human IgG: 1 µg

Scientific Data

SDS-PAGE View Larger

2 μg/lane of Recombinant Streptococcus equi subsp. zooepidemicus IgG endopeptidase IdeZ His-tag Protein (Catalog # 11572-IZ) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 35-39 kDa, under reducing conditions.

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: IdeZ

IdeZ from bacterial pathogen S. equi subspecies zooepidemicus, a group C streptococci common in animal infection, is a cysteine protease IgG endopeptidase that acts to ablate immune effector function during infection. As a homolog of the better-known IdeS/MAC from S. pyogenes, IdeZ also contains conserved critical residues that allow it to recognize and cleave at a single site below the hinge of IgG to produce F(ab')2 and Fc fragments with high specificity (1,2). However, IdeZ contains only a single cysteine and can cleave IgG more efficiently under non-reducing conditions (2). The bacterial pathogen is known to have a broad host range and since it is capable of cleaving IgG from multiple species it can cause pathogenesis in many animals such as strangles in horses (1), hemorrhagic pneumonia in dogs in kennels (3), and mastitis in ruminates on farms (4). S. zooepidemicus human infection has been reported from direct exposure to infected animals or contracted from infected animal products resulting in nephritis, hospitalization and death as IdeZ is capable of efficient cleavage of all human IgGs (5-8).  IdeZ has been reported as a tool in analytical strategies to characterize therapeutic antibodies (9,10) and to cleave proteins post-translationally modified with O-GlcNAc to enable immunoprecipitation analysis (11). IdeZ's broader specificity compared to IdeS means it can more effectively be used in animal model studies, for example due to its ability to more effectively cleave mouse IgG2a and IgG3. IdeZ has also been proposed as a target to develop vaccines to protect animals against group C streptococci (12). Finally, IdeZ has been used in the development of recombinant adeno-associated virus (AAV) vectors for the genetic therapy of rare diseases to eliminate already existing circulating neutralizing antibodies against recombinant AAVs (13). 

References
  1. Boksha, I.S. et. al. (2023) Biochemistry 88:731.
  2. Lannergard, J. and B. Guss (2006) FEMS Microbiol. Lett. 262:230.
  3. Priestnall, S. and K. Erles (2011) Vet. J. 188:142.
  4. Pisoni, G. et. al. (2009) J. Dairy Sci.92:943.
  5. Bradley, S.F. et. al. (1991) Rev. Infect. Diseases 13:270.
  6. Balter, S. et. al. (2000) Lancet. 355:1776.
  7. Abbott, Y. et. al. (2010) J. Med. Microbiol. 59:120.
  8. Pelkonen, S. et. al. (2013) Emerg. Infect. Dis. 19:1041.
  9. Bobaly, B. et. al. (2017) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.1060:325.
  10. Ruppen, I. et. al. (2023) J. Pharm. Biomed. Anal. 236:115743. 
  11. Machacek, M. et. al. (2020) Anal. Biochem. 611:114001.
  12. Velineni, S. and J.F. Timoney (2013) Vaccine. 31:4129.
  13. Elmore, Z.C. et. al. (2020) JCI Insight 5:e139881.

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