Recombinant Human Serpin E2/PN1 Protein, CF

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

Recombinant Human Serpin E2/PN1 Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <2 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Serpin E2/PN1 protein
  Met1-Pro397, with a C-terminal 10-His tag
• Accession #: NP_001130000
• N-terminal Sequence: Ser20
• Protein/Peptide Type: Recombinant Enzymes
• Gene: SERPINE2
• 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:
  • Inhibition Activity
• Theoretical MW: 43 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: 50 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 Sodium Acetate, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Serpin E2/PN1(rhSerpin E2) (Catalog # 2980-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Trypsin to 0.25 μg/mL in Assay Buffer and KEEP ON ICE.
  2. Prepare a curve of rhSerpin E2 (MW: 43,200 Da) in Assay Buffer. Make the following serial dilutions: 250, 100, 50, 30, 20, 10, 5, 2.5, and 0.5 nM.
  3. Combine equal volumes of diluted Trypsin and rhSerpin E2 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin without any rhSerpin E2.
  4. Incubate at room temperature for 30 minutes.
  5. Dilute reaction mixtures five fold in Assay Buffer.
  6. Dilute Substrate to 20 μM in Assay Buffer.
  7. Load 50 μL of the rhSerpin E2 curve in a plate, and start the reaction by adding 50 μL of 20 μM Substrate to wells.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC₅₀) for rhSerpin E2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin at each point may be determined using the following formula (if needed):

  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 MCA-Pro-Lys-OH (Bachem, Catalog # M-1975).

  Per Well:
  • Trypsin: 0.00125 μg
  • rhSerpin E2 curve: 12.5, 5, 2.5, 1.5, 1, 0.5, 0.25, 0.125, and 0.025 nM
  • Substrate: 10 μM

Notes

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

Alternate Names for Recombinant Human Serpin E2/PN1 Protein, CF

• GDN
• GdNPF
• GDNserine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 2
• glia-derived nexin
• glial-derived neurite promoting factor
• Peptidase inhibitor 7
• PI-7
• PI7DKFZp686A13110
• PN-1
• PN1nexin
• PNI
• Protease nexin 1
• Protease nexin I
• Proteinase Nexin 1
• Serpin E2
• serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitortype 1), member 2

Background

Serpin E2, also known as protease nexin I or glial-derived nexin (GDN), is a member of the Serpin superfamily of the serine protease inhibitors (1). Serpin E2 is a potent inhibitor of thrombin, plasmin and plasminogen activators (2). It is differentially expressed during neuronal differentiation and is able to transform human embryonic kidney cells into neuron-like cells (3). Its over‑expression in mice leads to progressive neuronal and motor dysfunction in these animals (4). It is also over‑expressed in the majority of pancreatic carcinoma as well as gastric and colorectal cancer samples whereas it is weakly expressed in all normal pancreas and chronic pancreatitis tissue samples (5). It plays an important role in controlling male fertility because its knockout male mice show a marked impairment in fertility from the onset of sexual maturity and its abnormal expression is found in the semen of men with seminal dysfunction (6). The deduced amino acid sequence of rhSerpin E2 is the same as that in NP_001130000, which predicts Arg329 in its 397 amino acid residues (7). An alternatively splice form predicts Thr-Gly at positions 329 and 330 in its 398 amino acid sequence (8-10).

1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
2. Rossignol, P. et al. (2004) J. Biol. Chem. 279:10346.
3. Lin, H.J. et al. (2005) Int. J. Dev. Neurosci. 23:9.
4. Meins, M. et al. (2001) J. Neurosci. 21:8830.
5. Buchholz, M. et al. (2003) Cancer Res. 63:4945.
6. Murer, V. et al. (2001) Proc. Natl. Acad. Sci. USA 98:3029.
7. Strausberg, R.L. et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899.
8. Sommer, J. et al. (1987) Biochemistry 26:6407.
9. Gloor, S. et al. (1986) Cell 47:687.
10. McGrogan, M. et al. (1988) Bio/Technology 6:172.

Publications for Serpin E2/PN1 (2980-PI)(6)

Publications using 2980-PI

• R Szabo, TH Bugge Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy PLoS ONE, 2018-04-04;13(4):e0194660. 2018-04-04 [PMID: 29617460] (Bioassay)
• The Involvement of Protease Nexin-1 (PN1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration Sci Rep, 2016-07-27;6(0):30563. 2016-07-27 [PMID: 27460424] (Bioassay, Human)
• Santoro A, Conde J, Scotece M, Abella V, Lois A, Lopez V, Pino J, Gomez R, Gomez-Reino J, Gualillo O SERPINE2 Inhibits IL-1alpha-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-kappaB/AP-1 Pathways. PLoS ONE, 2015-08-25;10(8):e0135979. 2015-08-25 [PMID: 26305372] (Bioassay, Human)
• Sales KU, Masedunskas A, Bey AL Matriptase initiates activation of epidermal pro-kallikrein and disease onset in a mouse model of Netherton syndrome. Nat. Genet., 2010-07-25;42(8):676-83. 2010-07-25 [PMID: 20657595] (Bioassay, Human)
• Myerburg MM, McKenna EE, Luke CJ, Frizzell RA, Kleyman TR, Pilewski JM Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis. Am. J. Physiol. Lung Cell Mol. Physiol., 2008-02-29;294(5):L932-41. 2008-02-29 [PMID: 18310226] (Bioassay, Human)
• List K, Currie B, Scharschmidt TC, Szabo R, Shireman J, Molinolo A, Cravatt BF, Segre J, Bugge TH Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice. J. Biol. Chem., 2007-10-16;282(50):36714-23. 2007-10-16 [PMID: 17940283] (Bioassay, Human)

Bioinformatics

• Gene Symbol: SERPINE2
• Uniprot:
  • NP_001130000()