Recombinant Human Serpin G1 His-tag Protein, CF

2 μg/lane of Recombinant Human Serpin G1 His-tag Protein (Catalog # 10951-PI) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 89-98 kDa.

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

Recombinant Human Serpin G1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit Recombinant Human Complement Component C1s (Catalog # 2060-SE) cleavage of a colorimetric peptide substrate, N-carbobenzyloxy-Lys-ThioBenzyl ester (Z-K-SBzl). The IC₅₀ is <5 nM, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Serpin G1/C1 Inhibitor protein
  Asn23-Ala500 with a C-terminal 6-His tag
• Accession #: P05155.2
• N-terminal Sequence: Asn23
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 54 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: 89-98 kDa, under 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Sodium Acetate and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, and 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
  • Recombinant Human Serpin G1 (Catalog # 10951-PI)
  • Recombinant Human Complement Component C1s (rhC1s) (Catalog # 2060-SE)
  • Substrate: L-Lys-SBzl (Bachem, Catalog # M-1300), 10 mM stock in DMSO
  • 5,5'-Dithobis(2-Nitrozenzoic acid) (DTNB) (Sigma, Catalog # D8130), 10 mM stock in DMSO
  • 96-well Clear Plate (Catalog # DY990)
  • Fluorescent Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhC1s to 2 µg/mL in Assay Buffer.
  2. Prepare a curve of rhSerpin G1 (MW: 54,000 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 100, 50, 25, 12.5, 4, 0.8, and 0.2 nM.
  3. Combine 25 µL of diluted rhC1s and 25 µL of rhSerpin G1 at each concentration of the curve. Include a Control containing 25 µL of Assay Buffer and 25 µL of diluted rhC1s.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Dilute the mixtures 5-fold by adding 200 µL of Assay Buffer to each reaction and control vial.
  6. Dilute the Substrate to 200 µM with Assay Buffer containing 200 µM DTNB.
  7. Load into plate 50 µL of the diluted incubated mixtures and start the reaction by adding 50 µL of 200 µM Substrate/200 µM DTNB mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate/ µM DTNB mixture.
  8. Read at an absorbance wavelength of 405 nm in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC50) value for rhSerpin G1 by plotting OD/min (or specific activity) versus concentration with 4-PL fitting.
  10. The specific activity for rhC1s at each point may be determined using the following formula (if needed):

  Specific Activity(pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / ((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 the many spectrophotometers is in mOD

  Per Well:
  • rhSerpin G1 curve: 100, 50, 25, 5, 2.5, 1.25, 0.625, 0.2, 0.04, and 0.01 nM
  • rhC1s: 0.01 µg
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

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

Alternate Names for Recombinant Human Serpin G1 His-tag Protein, CF

• C1 Inhibitor
• C1IN
• C1INH
• C1NH
• HAE1
• HAE2
• Serpin G1
• SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1

Background

C1-inhibitor (C1-INH), also known as Serpin G1, is a 478 amino acid acute phase plasma glycoprotein member of the serpin superfamily of serine protease inhibitors.  C1 inhibitor, like many other serpin family members, contains a highly conserved C-terminal serpin domain with a reactive center loop that protrudes to present a peptide for proteolytic attack (1). Binding disrupts the protease active site and traps it in a covalent complex with C1-inhibitor resulting in inactivation of the protease. C1 inhibitor also contains a highly glycosylated N- terminus and a unique C-terminal tail that has been suggested to serve as a barrier for transition to an inactive latent form (2). C1 inhibitor is synthesized and secreted primarily by hepatocytes but also by other cells such as monocytes, fibroblasts, and macrophages (1). C1 inhibitor is the sole physiological inhibitor of activated classical complement pathway serine proteases C1r and C1s (3). In addition, C1 inhibitor can inhibit multiple other serine proteases including plasma kallikrein and coagulation factor XIIa from the contact activation system, MASP-1 and MASP-2 proteases from the mannose binding lectin pathway, factor XIa and thrombin from the coagulation system, and plasmin and tissue plasminogen activator from the fibrinolytic system (3). Serpin G1 deficiency, whether caused by deletions, mutations, or inactivation, results in an autosomal dominantly inheritable potentially lethal disease, hereditary angioedema (HAE) (1). Some mutations in the SerpinG1 gene are also associated with age-related macular degeneration (4). Given the role C1-inhibitor plays as a potent anti-inflammatory agent, it has been developed for clinical use to treat HAE (1, 5) and considered for treatment of several other conditions including sepsis, acute myocardial infarction, brain injury, transplantation and COVID19 (6-11).

1. Karnaukhova, E. (2013) J. Hematol. Thromb. Dis. 1:3.
2. Beinrohr, L. et al. (2007) J. Biol. Chem. 282:21100.
3. Davis, A.E. III (2010) Thromb. Haemost. 104:886.
4. Ennis, S. et al. (2008) Lancet 372:1828.
5. Cicardi, M and A. Zanichelli (2010) Drugs Today. 46:867.
6. Caliezi, C. et al. (2000) Pharmacol. Rev. 52:91.
7. Thielmann, M. et al. (2006) Eur. J. Cardiothorac. Surg. 30:285.
8. Singer, M. and Jones, A.M. (2011) Crit. Care 15:203.
9. Berger, M. et al. (2016) Transplantation 100:1415.
10. Weiss, E. et al. (2020) Clin. Exp. Emerg. Med. 7:87.
11. Adesanya, T.M.A. et al. (2021) J. Clin. Immunol. 41:729.

Bioinformatics

• Uniprot:
  • P05155.2()