Recombinant Human R-spondin 3, Animal-Free Protein

Cells transfected with reporter TOP-FLASH are treated in triplicate with increasing concentration of R-spondin 3 in the presence of Wnt-conditioned media (1:8 dilution). RSP1 enhances Wnt-beta catenin signaling in HEK239T cells. Cells are grown overnight, and luciferase activity is measured and normalized.EC50 = 14 ng/ml (0.8 nM).

R-spondin 3 bioactive domain migrates at 17 kDa in non-reducing (-beta ME) conditions and upon reduction (+ beta ME).Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+ beta -mercaptoethanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human R-spondin 3, Animal-Free Protein Summary

• Details of Functionality: No significant difference between EC₅₀ of reference and test lots
• Source: E. coli-derived human R-Spondin 3 protein
• Accession #: Q9BXY4.1
• Protein/Peptide Type: Animal-Free Recombinant Proteins
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 17 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: Monomeric R-spondin 3 protein only

Packaging, Storage & Formulations

• Storage: Store lyophilized protein between -20 and -80 °C until the date of expiry. Avoid freeze-thaw cycles.
• Buffer: Lyophilized from acetonitrile/TFA
• Reconstitution Instructions: Resuspend in 10 mM HCl at >100 µg/ml, prepare single use aliquots, add carrier protein if desired.

Notes

The above product was manufactured, tested and released by R&D System's contract manufacturer, Qkine Ltd, at 1 Murdoch House, Cambridge, UK, CB5 8HW. The product is for research use only and not for the diagnostic or theraputic use.

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

Alternate Names for Recombinant Human R-spondin 3, Animal-Free Protein

• Cristin 1
• CRISTIN1
• FLJ14440
• hPWTSR
• hRspo3
• Protein with TSP type-1 repeat
• PWTSR
• Roof plate-specific spondin-3
• RSPO3
• R-spondin 3 homolog (Xenopus laevis)
• RSpondin 3
• R-Spondin 3
• R-spondin-3
• Thrombospondin type-1 domain-containing protein 2
• thrombospondin, type I, domain containing 2
• THSD2

Background

R-Spondin 3 (RSPO3, roof plate-specific spondin 3), also called cysteine-rich and single thrombospondin domain containing-1 (Cristin 1), is an ~31 kDa secreted protein that shares ~40% amino acid (aa) identity with the other three R-Spondin family members (1, 2). All are positive modulators of Wnt/ beta -catenin signaling, but each has a distinct expression pattern (1-4). Like other R-spondins,R-Spondin 3 contains two adjacent cysteine-rich furin-like domains (aa 35-135) with one potential N-glycosylation site (aa 36), followed by a thrombospondin (TSP-1) motif (aa 147-207) and a region rich in basic residues (aa 211-269). Only the furin-like domains are needed for beta -catenin stabilization (2). Within aa 21-209, human R-Spondin 3 shares 93%, 92%, 97%, 96% and 92% aa identity with mouse, rat, equine, bovine and canine R-Spondin 3, respectively. Potential isoforms of 279 and 297 aa diverge at aa 210 and 276, respectively (5). Mouse R-Spondin 3 is critical for development of the placental labyrinthine layer, probably by promoting VEGF expression and thus vascular development (6, 7). It is also essential for expression of the placenta-specific transcription factor, Gcm1. In the mouse embryo, R-Spondin 3 is often expressed by or located near endothelial cells (6). It is found in the roof plate, tail, somites, otic vesicles, cephalic mesoderm, truncus arteriosus, atrioventricular canal of the developing heart, and strongly but transiently in developing limbs (4, 7). R-Spondins regulate Wnt/ beta -catenin by competing with the Wnt antagonist DKK-1 for binding to the Wnt co-receptors LRP-6 and Kremen, reducing their DKK-1-mediated internalization (8, 9). Reports differ on whether R-Spondins bind LRP-6 directly (8-10). R-Spondin 3 has also been identified as an oncogene (11).

1. Chen, J-Z. et al. (2002) Mol. Biol. Rep. 29:287.
2. Kim, K.-A. et al. (2008) Mol. Biol. Cell 19:2588.
3. Hendrickx, M. and L. Leyns (2008) Develop. Growth Differ. 50:229.
4. Nam, J.-S. et al. (2007) Gene Expr. Patterns 7:306.
5. Entrez Accession # EAW48114 and EAW48116.
6. Kazanskaya, O. et al. (2008) Development 135:3655.
7. Aoki, M. et al. (2007) Dev. Biol. 301:218.
8. Binnerts, M.E. et al. (2007) Proc. Natl. Acad. Sci. USA 104:14700.
9. Nam, J.-S. et al. (2006) J. Biol. Chem. 281:13247.
10. Wei, Q. et al. (2007) J. Biol. Chem. 282:15903.
11. Theodorou, V. et al. (2007) Nat. Genet. 6:759.

Bioinformatics

• Uniprot:
  • Q9BXY4.1()