Recombinant human R-spondin 1 protein

Summary

• High purity recombinant human R-spondin 1 protein (Uniprot: Q2MKA7)
• >98%, by SDS-PAGE quantitative densitometry
• 13 kDa
• Expressed in E. coli
• Animal origin-free (AOF) and carrier protein-free.
• Manufactured in Qkine's Cambridge, UK laboratories
• Lyophilized from acetonitrile, TFA
• Resuspend in 10mM HCl at >100 µg/ml (provided with protein and free of charge), prepare single use aliquots, add carrier protein if desired and store frozen at -20°C or -80°C

Featured applications

• Pancreatic tumor organoid culture
• Maintenance of urine-derived stem cells

Purity

R-spondin 1 (Qk006) has be tested at 100 ng/ml – 500 ng/ml, we would recommend using 100 ng/ml – 1 µg/ml for initial testing and optimisation with culture. It is highly recommended to do a dose response to optimize concentration due to varying culture conditions across different labs.

Qkine FGF-4 bioactivity is comparable to an alternative supplier protein

Quantitative luciferase assay with Qkine FGF-4 (Qk004, green) and alternative supplier FGF-4 (Supplier B, black). Cells were treated in triplicate with a serial dilution of FGF-4 for 3 hours. Firefly luciferase activity was measured and normalized to control Renilla luciferase activity.

Protein background

R-spondin 1 (RSPO-1) is a secreted protein of the RSPO family that regulates the canonical Wnt signaling pathway [1,2]. The Wnt pathway is a highly conserved signaling pathway that is essential for various cellular processes, including cell proliferation, differentiation, and migration. This growth factor plays a crucial role in embryonic development and patterning, particularly in the development of the gastrointestinal tract, neural tube, skin, and limbs [3]. It is involved in the maintenance of stem cells and can enhance the self-renewal capacity of stem cells, promoting tissue regeneration and repair [4]. It is also involved in the sex determination and the development and maintenance of the female sex characteristics in particular the differentiation and maintenance of the ovarian tissues [5].

RSPO1 proteins are generally characterized by a core domain known as the thrombospondin type 1 (TSP1) domain, which is responsible for their signaling activity. This protein is comprised of 263 amino acids and structured as two cysteine-rich, furin-like domains [3,6]. It acts as an agonist (activator) of the Wnt signaling pathway by binding to the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-6, leading to the stabilization of β-catenin, a key component of the pathway [7].

In cell culture, this recombinant protein is used extensively for the maintenance of self-renewal and expansion of stem cells. It is also used for the culture of intestinal, hematopoietic, and epithelial stem cells [2,7,8]. Also, the culture of stem cell-derived organoids such as intestinal, pancreas liver, colon, and stomach organoids benefit from the inclusion of  RSPO1 in the media [7,9,10].

Dysregulation of the Wnt signaling pathway, including the overexpression of R-spondin 1, has been associated with tumor growth and progression in various cancers such as colon and ovarian cancer [11,12]. Research is ongoing on the role of R-spondin 1 in various biological processes and its potential as a therapeutic target for cancer treatment. Given its role in stem cell maintenance and tissue regeneration,  RSPO1 and related proteins have also been explored as potential therapeutic targets for regenerative medicine.

Background references

1. K.-A. Kim et al., ‘R-Spondin family members regulate the Wnt pathway by a common mechanism’, Mol. Biol. Cell, vol. 19, no. 6, pp. 2588–2596, Jun. 2008, doi: 10.1091/mbc.e08-02-0187.
2. W. de Lau, W. C. Peng, P. Gros, and H. Clevers, ‘The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength’, Genes Dev., vol. 28, no. 4, pp. 305–316, Feb. 2014, doi: 10.1101/gad.235473.113.
3. A. Glinka et al., ‘LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling’, EMBO Rep., vol. 12, no. 10, pp. 1055–1061, Sep. 2011, doi: 10.1038/embor.2011.175.
4. K. S. Yan et al., ‘Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal’, Nature, vol. 545, no. 7653, Art. no. 7653, May 2017, doi: 10.1038/nature22313.
5. K. Tomizuka et al., ‘R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling’, Hum. Mol. Genet., vol. 17, no. 9, pp. 1278–1291, May 2008, doi: 10.1093/hmg/ddn036.
6. W. B. de Lau, B. Snel, and H. C. Clevers, ‘The R-spondin protein family’, Genome Biol., vol. 13, no. 3, p. 242, Mar. 2012, doi: 10.1186/gb-2012-13-3-242.
7. W. de Lau et al., ‘Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling’, Nature, vol. 476, no. 7360, Art. no. 7360, Aug. 2011, doi: 10.1038/nature10337.
8. J. R. Genthe and W. K. Clements, ‘R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways’, Development, vol. 144, no. 4, pp. 590–600, Feb. 2017, doi: 10.1242/dev.139956.
9. L. Broutier et al., ‘Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation’, Nat. Protoc., vol. 11, no. 9, Art. no. 9, Sep. 2016, doi: 10.1038/nprot.2016.097.
10. L. Broutier et al., ‘Human primary liver cancer–derived organoid cultures for disease modeling and drug screening’, Nat. Med., vol. 23, no. 12, Art. no. 12, Dec. 2017, doi: 10.1038/nm.4438.
11. S. Seshagiri et al., ‘Recurrent R-spondin fusions in colon cancer’, Nature, vol. 488, no. 7413, Art. no. 7413, Aug. 2012, doi: 10.1038/nature11282.
12. E. J. ter Steege and E. R. M. Bakker, ‘The role of R-spondin proteins in cancer biology’, Oncogene, vol. 40, no. 47, Art. no. 47, Nov. 2021, doi: 10.1038/s41388-021-02059-y.

Publications using recombinant human R-spondin 1 protein (Qk006)

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro
In Experimental Dermatology on 28 April 2023 by Agarwal, R. et al.
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In Cancer Research and Treatment on 12 June 2023 by Choi, W et al.
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The effect of extracellular matrix on the precision medicine utility of pancreatic cancer patient–derived organoids.
In JCI Insight on 5 December 2023 by Lumibao, J.C., Okhovat, S.R, et al.
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In Cells Reports Medicine on 8 March 2024 by Ku, B., Eisenbarth, D., et al.
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