Recombinant Human R-spondin 1, Animal-Free Protein

R-spondin 1 activity is determined using the Wnt-responsive firefly luciferase reporter assay as it enhances Wnt-beta catenin signaling in HEK239T cells. HEK293T cells transfected with reporter TOP-FLASH are treated in triplicate with increasing concentration of R-spondin 1 (diluted in DMEM with 0.5 % of FCS), in the presence of Wnt-conditioned media (1:8 dilution). Cells are grown overnight, and luciferase activity is measured and normalized.EC50 = 75.5 ng/ml (5.8 nM).

RSPO1 migrates as a single band at 16 kDa in non-reducing (NR) and 13 kDa in reducing (R) conditions.Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+ beta -mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.

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

Recombinant Human R-spondin 1, Animal-Free Protein Summary

• Details of Functionality: No significant difference between EC 50 of reference and test lots
• Source: E. coli-derived human R-Spondin 1 protein
• Accession #: Q2MKA7.1
• 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: 13 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 1 protein only

Packaging, Storage & Formulations

• Storage: Store lyophilized protein between -20 and -80 degreesC until the date of expiry. Avoid freeze-thaw cycles.
• Buffer: Lyophilized from acetonitrile/TFA
• Reconstitution Instructions: Resuspend in 10mM 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 1, Animal-Free Protein

• Cristin 3
• CRISTIN3
• FLJ40906Roof plate-specific spondin-1
• HRspo1
• roof plate-specific spondin
• RSPO
• RSPO1
• RSpondin 1
• R-Spondin 1
• R-spondin homolog (Xenopus laevis)
• RSPONDIN
• R-spondin1
• R-spondin-1

Background

R-Spondin 1 (RSPO1, Roof plate-specific Spondin 1), also known as cysteine-rich and single thrombospondin domain containing protein 3 (Cristin 3), is a 27 kDa secreted protein that shares ~40% amino acid (aa) identity with three other R-Spondin family members (1, 2). All R-Spondins regulate Wnt/ beta-Catenin signaling but have distinct expression patterns (1-3). R-Spondin 1 competes with the Wnt antagonist DKK-1 for binding to the Wnt co-receptors, Kremen and LRP-6, reducing their DKK-1-mediated internalization (4). However, reports are mixed on whether R-Spondin 1 binds LRP-6 directly (4-6). R-Spondin 1 is expressed in early development at the roof plate boundary and is thought to contribute to dorsal neural tube development (3, 7). In humans, rare disruptions of the R-Spondin 1 gene are associated with tendencies for XX sex reversal (phenotypic male) or hermaphroditism, indicating a role for R-Spondin 1 in gender-specific differentiation (7, 8). Mutations in R-Spondin 1 are also linked with palmoplantar keratoderma, abnormal thickening of the skin on the palms of the hands and soles of the feet (7, 8). Postnatally, R-Spondin 1 is expressed by neuroendocrine cells in the intestine, adrenal gland and pancreas, and by epithelia in kidney and prostate (9). Injection of recombinant R-Spondin 1 in mice causes activation of beta-catenin and proliferation of intestinal crypt epithelial cells, and ameliorates experimental colitis (9, 10). Interest in R-Spondin 1 as a cell culture supplement has grown with the expansion of the organoid field. R-Spondin 1 is widely used in organoid cell culture workflows as a vital component that promotes both growth and survival of 3D organoids (11). Structurally similar to other R-Spondins, R-Spondin 1 contains two adjacent cysteine-rich furin-like domains (aa 34-135) with one potential N-glycosylation site, followed by a thrombospondin (TSP-1) motif (aa 147-207) and a region rich in basic residues (aa 211-263). Only the furin-like domains are needed for beta-catenin stabilization (2, 12). A putative nuclear localization signal at the C-terminus may allow some expression in the nucleus (13). Potential isoforms of 200 and 236 aa have an alternate, shorter N-terminus or are missing aa 146-208, respectively (14). Over aa 21-263, human R-Spondin 1 shares 89%, 87%, 92%, 91%, 91% and 89% aa identity with mouse, rat, horse, dog, goat, and cow RSPO-1, respectively.

1. Chen, J-Z. et al. (2002) Mol. Biol. Rep. 29:287.
2. Kim, K.-A. et al. (2006) Cell Cycle 5:23.
3. Nam, J.-S. et al. (2007) Gene Expr. Patterns 7:306.
4. Binnerts, M.E. et al. (2007) Proc. Natl. Acad. Sci. USA 104:14700.
5. Nam, J.-S. et al. (2006) J. Biol. Chem. 281:13247.
6. Wei, Q. et al. (2007) J. Biol. Chem. 282:15903.
7. Kamata, T. et al. (2004) Biochim. Biophys. Acta 1676:51.
8. Parma, P. et al. (2006) Nat. Genet. 38:1304.
9. Kim, K.-A. et al. (2005) Science 309:1256.
10. Zhao, J. et al. (2007) Gastroenterology 132:1331.
11. Drost and Clevers. (2018) Nature Reviews Cancer 18:407.
12. Kazanskaya, O. et al. (2004) Dev. Cell 7:525.
13. Tomaselli, S. et al. (2008) Hum. Mutat. 29:220.
14. UniProt # Q2MKA7.

Bioinformatics

• Uniprot:
  • Q2MKA7.1()