Recombinant Human Complement Component C1rLP His Protein, CF

Recombinant Human Complement Component C1rLP HA-tag His-tag (Catalog # 11614-CP) is measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Gly-Arg-ThioBenzyl ester (Z-GR-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB).

2 μg/lane of Recombinant Human Complement Component C1rLP HA-tag His-tag Protein (Catalog # 11614-CP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 70‑76 kDa, under reducing conditions.

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

Recombinant Human Complement Component C1rLP His Protein, CF Summary

• Additional Information: HA-tag His-tag
• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Gly-Arg-ThioBenzyl ester (Z-GR-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 >30 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Complement Component C1rLP protein
  Cys36-Asp487 with N-terminal HA (YPYDVPDYA) and 6-His tags
• Accession #: NP_057630.1
• N-terminal Sequence: Tyr
• 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:
  • Binding Activity2
• Theoretical MW: 52 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: 70-76 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 opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Recombinant Human Complement Component C1rLP (rhC1rLP) (Catalog # 11614-CP)
  • Substrate: Z-GR-SBzl,10 mM stock in DMSO
  • 5,5'Dithio-­bis (2-­nitrobenzoic acid) (DTNB), 10 mM stock in DMSO
  • Clear 96-well plate
  • Plate Reader with Absorbance Read Capability
  1. Dilute rhC1rLP to 40 ng/µL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 400 µM of Z-GR-SBzl and 200 µM of DTNB in Assay Buffer.
  3. In a plate, load 50 µL of 40 ng/µL rhC1rLP and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read plate at 405 nm (absorbance) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  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 of 0.320 cm
  Per Well:
  • rhC1rLP: 2 µg
  • Z-GR-SBzl: 200 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 Complement Component C1rLP His Protein, CF

• C1RL
• C1RL1
• C1RL1complement C1r subcomponent-like protein
• C1r-like protein
• C1r-like serine protease analog protein
• C1r-LPcomplement C1r-like proteinase
• C1RLPEC 3.4.21.-
• CLSPa
• complement component 1, r subcomponent-like
• Complement Component C1rLP
• EC 3.4.21
• EC 3.4.21.41

Background

Recombinant human Complement C1r subcomponent-like protein (C1rLP), also known as C1r-like serine protease analog protein (CLSPa) is a 487 residue glycosylated, secreted serine peptidase-like protein (1, 2).  It contains an N-terminal signal peptide, a CUB domain for substrate and protein-protein interaction, a truncated complement control protein module, and a trypsin-like serine protease domain with a characteristic catalytic triad but lacks an activation consensus peptide and an EGF domain found in homologous complement cascade proteases like C1r protein (1, 2). It is broadly expressed and secreted in tissues and abundantly found in liver, kidney, and myeloid cells where significant expression of serine proteases typically occurs (1). C1rLP has esterolytic activity on peptide thioesters with arginine at the P1 position but lower catalytic efficiency compared to C1r (2). It was reported to mediate cleavage of haptoglobin (3) and subsequently proposed to be of use in hemoglobin-binding therapeutics (4). Complement cascade serine proteases such as C1r play a role in innate immunity but the role of C1rLP in complement-mediated function is unclear as both activation and inhibition roles have been described in the literature (1, 2). C1rLP has been shown to be a pathological marker in immune microenvironments including within acute myocardial infarction and COVID19 as well as several types of cancer such as colon, leukemia, brain, endometrial, and melanoma (5-12). As a pathological marker, it has also been proposed as a potential therapeutic target for the treatment of these diseases (12).

1. Lin, N. et al. (2004) Biochem. Biophys. Res. Commun. 321:329.
2. Ligoudistianou, C. et al. (2005) Biochem. J. 387:165.
3. Wicher, K.B. et al. (2004) Proc. Natl. Acad. Sci. 101:14390.
4. Schaer, C.A. et al. (2018) BMC Biotechnol. 18:15. 
5. Liu, X. et al. (2021) ACS Omega 6:7951.
6. Yang, S. et al. (2022) Front. Oncol. 12:934928.
7. Chen J. et al. (2023) Aging 15:4444.
8. Njoku K. et al. (2023) Br. J. Cancer 128:1723.
9. Yoon, H.G. et al. (2023) PLoS One 18:e0295061.
10. Beck. H.C. et al. (2024) Int. J. Mol. Sci. 25:2638.
11. Liu, G. et al. (2024) Heliyon 11:e32337.
12. Zhang, M. et al. (2024) Heliyon 10:e30616.

Bioinformatics

• Uniprot:
  • NP_057630.1()