Recombinant Human Granzyme H Protein, CF

R&D Systems | Catalog # 1377-SE

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human Granzyme H Protein (1377-SE)
  • 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

Source

NS0

Accession Number

P20718

Structure / Form

Pro form

Applications

Enzyme Activity
View all Granzyme H Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Granzyme H protein
Glu19-Leu246, with a C-terminal 10-His tag
Accession # P20718

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Glu19

Predicted Molecular Mass

27 kDa

SDS-PAGE

Multiple bands between 38-40 kDa, reducing conditions

Activity

Measured by its ability to cleave a peptide substrate, Succinyl-Phe-Leu-Phe-ThioBenzyl ester (Suc-FLF-SBzl), in the presence of 5,5’-Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468.
The specific activity is >5,000 pmol/min/µg, as measured under the described conditions.

Formulation, Preparation, and Storage

1377-SE
Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Brij-35.
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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: Granzyme H

Granzyme H is a member of the granzyme family of serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1, 2). Granzyme H’s functions are largely unknown. The more abundant expression of Granzyme H than Granzyme B in NK cells suggests that Granzyme H may complement the pro-apoptotic function of Granzyme B in this cell type (3). Human Granzyme H shows the highest amino acid identity (71%) to mouse Granzyme C (4). Human Granzyme H is synthesized as a precursor (246 residues) with a signal peptide (residues 1-18), a propeptide (residues 19-20) and a mature chain (residues 21-246) (5-7). The pro-enzyme is expressed and purified. After being activated by active cathepsin C, rhGranzyme H cleaves a thioester substrate, which was described previously (8).

References

  1. Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
  2. Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
  3. Sedelies, K.A. et al. (2004) J. Biol. Chem. 279:26581.
  4. Sattar, R. et al. (2003) Biochem. Biophys. Res. Comm. 308:726.
  5. Meier, M. et al. (1990) Biochemistry 29:4042.
  6. Haddad, P. et al. (1991) Int. Immunol. 3:57.
  7. Klein, J.L. et al. (1990) Tissue Antigens 35:220.
  8. Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468.
    9.  

Alternate Names

CCP-X, CGL2, CTSGL2, GrzH, GZMH

Entrez Gene IDs

2999 (Human)

Gene Symbol

GZMH

UniProt

P20718

Additional Granzyme H Products

Product Documents for Recombinant Human Granzyme H Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Granzyme H Protein, CF

For research use only

Citations for Recombinant Human Granzyme H Protein, CF

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Protocols

View specific protocols for Recombinant Human Granzyme H Protein, CF (1377-SE):

Materials
  • Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
  • Assay Buffer: 50 mM Tris, 0.5 M NaCl, pH 9.0
  • Recombinant Human Granzyme H (rhGranzyme H) (Catalog # 1377-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • Substrate: Succinyl-Phe-Leu-Phe ThioBenzyl ester (Suc-FLF-SBzl) (Bachem, Catalog # M-1740), 10 mM stock in DMSO
  • 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Activate rhGranzyme H at 100 µg/mL with 21 µg/mL of rmCathepsin C in Activation Buffer.
  2. Incubate reaction at 37 °C for 2 hours.
  3. Dilute activated rhGranzyme H to 0.4 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM containing 200 µM of DTNB in Assay Buffer.
  5. In a plate load 50 µL of 0.4 ng/µL rhGranzyme H, and start the reaction by adding 50 µL of 200 µM Substrate mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate mixture.
  6. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  7. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/M
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 13260 M-1cm-1 
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:

  • rhGranzyme H: 0.02 µg
  • DTNB: 100 µM
  • Substrate: 100 µM

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