Recombinant Human Neutrophil Elastase/ELA2 Protein, CF

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

Recombinant Human Neutrophil Elastase/ELA2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, MeOSuc-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (MeOSuc-AAPV-AMC). The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Neutrophil Elastase/ELA2 protein
  Ser28-Asn252, with a C-terminal 10-His tag
• Accession #: P08246
• N-terminal Sequence: Ser28
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 25 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: 29-39 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
  • Assay Buffer: 50 mM Tris, 1 M NaCl, 0.05% (w/v) Brij-35, pH 7.5
  • Recombinant Human Elastase/ELA2 (rhELA2) (Catalog # 9167-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • Substrate: MEOSUC-Ala-Ala-Pro-Val-AMC (Bachem, Catalog # I-1270), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhELA2 to 50 µg/mL in Activation Buffer containing 50 µg/mL rmCathepsin C.
  2. Incubate for 2 hours at 37 °C to activate rhELA2.
  3. Dilute active rhELA2 to 1 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. Load into a plate 50 µL of 1 ng/µL rhELA2, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank.
  **Derived using calibration standard 7-amino, 4-Methly Coumarin (Sigma, Catalog # A9891).

  Per Well:
  • rhELA2: 0.05 µg
  • 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 Neutrophil Elastase/ELA2 Protein, CF

• Bone marrow serine protease
• EC 3.4.21
• EC 3.4.21.37
• ELA2
• ELA2granulocyte-derived elastase
• ELANE
• elastase 2, neutrophil
• elastase, neutrophil expressed
• Elastase-2
• GE
• HLEelastase-2
• HNE
• Human leukocyte elastase
• Leukocyte Elastase
• Medullasin
• NE
• Neutrophil Elastase
• PMN elastase
• PMN-E
• polymorphonuclear elastase
• SCN1

Background

Neutrophil Elastase (ELA2, ELANE), also known as HNE, is a chymotrypsin family serine protease that plays a key role in pathogen clearance (1-3). It is expressed by promyelocytes and stored in the intracellular azurophilic granules of polymorphonuclear leukocytes (PMN) (4). These granules fuse with phagosomes, enabling Neutrophil Elastase to participate in the digestion and killing of endocytosed microbes. The enzyme is released by activated neutrophils at sites of inflammation, and it can remain associated with the cell surface or function as a component of neutrophil extracellular nets (NETs) which trap and kill microbial pathogens (5, 6). It also can degrade multiple extracellular matrix proteins including Elastin and Fibronectin (5). In the lung, this activity contributes to pathology in emphysema, cystic fibrosis, and adult respiratory distress syndrome (ARDS) (1). Neutrophil Elastase can be inhibited by Serpin A1/alpha 1-Antitrypsin, SLPI, Serpin B1, and
Trappin-2/Elafin (7-11). Its activity in the lung is increased by exposure to tobacco smoke which inactivates Serpin A1 through methionine oxidation (12). Mature human Neutrophil Elastase shares 73% amino acid sequence identity with mouse and rat Neutrophil Elastase (13, 14). Multiple mutations in the human ELANE gene are causative of severe congenital and cyclic neutropenias (15).

1. Korkmaz, B. et al. (2010) Pharmacol. Rev. 62:726.
2. Stein, R.L. et al. (1987) Biochemistry 26:1301.
3. Bachovchin, W.W. (1986) Biochemistry 25:7751.
4. Garwicz, D. et al. (2005) Haematologica 90:38.
5. Owen, C.A. et al. (1995) J. Cell Biol. 131:775.
6. Stephan, A. and M. Fabri (2015) Exp. Dermatol. 24:161.
7. Carrell, R.W. et al. (1982) Nature 298:329.
8. Rice, W.G. and S.J. Weiss (1990) Science 249:178.
9. Thompson, R.C. et al. (1986) Proc. Natl. Acad. Sci. USA 83:6692.
10. Cooley, J. et al. (2001) Biochemistry 40:15762.
11. Wiedow, O. et al. (1990) J. Biol. Chem. 265:14791.
12. Taggart, C. et al. (2000) J. Biol. Chem. 275:27258.
13. Sinha, S. et al. (1987) Proc. Natl. Acad. Sci. USA 84:2228.
14. Okano, K. et al. (1987) J. Biochem. 102:13.
15. Makaryan, V. et al. (2015) Curr. Opin. Hematol. 22:3.

Publications for Neutrophil Elastase/ELA2 (9167-SE)(3)

Publications using 9167-SE

• P Cheung, S Schaffert, SE Chang, M Dvorak, M Donato, C Macaubas, MH Foecke, TM Li, L Zhang, JP Coan, GS Schulert, AA Grom, LA Henderson, PA Nigrovic, JE Elias, O Gozani, ED Mellins, P Khatri, PJ Utz, AJ Kuo Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation Nature Immunology, 2021-05-20;22(6):711-722. 2021-05-20 [PMID: 34017121] (Bioassay, Human)
• Z Li, D Peirasmaki, S Svärd, M Åbrink The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardiaintestinalis Cells, 2020-04-09;9(4):. 2020-04-09 [PMID: 32283818] (Bioassay, Human)
• J Guo, J Tu, Y Hu, G Song, Z Yin Cathepsin G cleaves and activates IL-36? and promotes the inflammation of psoriasis Drug Des Devel Ther, 2019-02-08;13(0):581-588. 2019-02-08 [PMID: 30804664] (Bioassay, Human)

Bioinformatics

• Uniprot:
  • P08246()