Recombinant Human Granzyme H Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Granzyme H Protein, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived human Granzyme H protein
  Glu19-Leu246, with a C-terminal 10-His tag
  Accession # P20718
• Accession #: P20718
• N-terminal Sequence: Glu19
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GZMH
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 27 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: Multiple bands between 38-40 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Granzyme H Protein, CF

• Cathepsin G-like 2
• CCP-X
• CCP-Xcytotoxic T-lymphocyte-associated serine esterase 1
• CGL2
• CGL-2
• CSP-Ccytotoxin serine protease-C
• CTLA1
• CTSGL2
• CTSGL2granzyme H
• Cytotoxic serine protease C
• Cytotoxic T-lymphocyte proteinase
• EC 3.4.21
• EC 3.4.21.-
• EC 3.4.21.79
• granzyme H (cathepsin G-like 2, protein h-CCPX)
• Granzyme H
• GrzH
• GZMH

Background

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).

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.
   
 

Publications for Granzyme H (1377-SE)(2)

Publications using 1377-SE

• BM Larimer, E Wehrenberg, F Dubois, A Mehta, T Kalomeris, K Flaherty, G Boland, U Mahmood Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response Cancer Res., 2017-05-01;77(9):2318-2327. 2017-05-01 [PMID: 28461564] (Enzyme Assay)
• Fellows E, Gil-Parrado S, Jenne D, Kurschus F Natural killer cell-derived human granzyme H induces an alternative, caspase-independent cell-death program. Blood, 2007-04-04;110(2):544-52. 2007-04-04 [PMID: 17409270] (Enzyme Assay)

Bioinformatics

• Gene Symbol: GZMH
• Uniprot:
  • P20718()