Recombinant human R-spondin 1 LR5 protein

Summary

• High purity recombinant human R-spondin 1 LR5 protein. Binds with high affinity to LGR5 receptor.
• 13 kDa
• >98%, by SDS-PAGE quantitative densitometry
• Expressed in E. coli
• Animal origin-free (AOF) and carrier protein-free
• Manufactured in our Cambridge, UK laboratories
• Lyophilized from acetonitrile, TFA
• Resuspend in 10 mM HCl (Reconstitution solution A) at >50 µg/ml, add carrier protein if desired, prepare single-use aliquots and store frozen at -20 °C (short-term) or -80 °C (long-term)

Featured applications

• Intestinal organoid culture
• Tumor organoid culture

Bioactivity

R-spondin 1 enhances Wnt signalling in TOP-Flash reporter assay. HEK293T cells transfected with Wnt-responsive firefly luciferase reporter TOP-FLASH are treated with increasing concentration of Qk031 R-spondin 1 LR5 (diluted in DMEM with 0.5 % of FCS), in the presence of Wnt-conditioned media (1:8 dilution), in triplicate. Cells are grown overnight and luciferase activity measured by luminescence. Data from Qk031 lot #104286. EC50 = 476 ng/ml (38 nM).  The higher EC50 than observed with wild-type R-spondin 1 Qk006 (72.7 ng/ml, 6nM) is due to the receptor population on HEK293 cells.

Purity

R-spondin 1 LR5 migrates as a single band at 16 kDa in non-reducing (NR) and 13 kDa in reducing (R) conditions. Purified recombinant protein (1 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250. Data from Qk031 lot #104286.

Qk031 is a specialized form of R-spondin 1 that specifically binds to the LGR5 receptor, which was developed in the lab of Marc de la Roche (University of Cambridge).  R-spondin 1 (Qk006) is a ligand for the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-6. Binding of R-spondin 1 to its coreceptors, LGR4-6 and ZNRF3, releases the inhibition ZNRF3 imposes on Wnt signalling.

In epithelial tissues LGR5 marks the stem cell population. This engineered form, R-spondin1 LR5, activates Wnt signalling only in the LGR5+ stem cell population.

Protein background

Human R-spondin 1 (RSPO-1) protein is a secreted activator of the canonical Wnt signaling pathway. A ligand for the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-6 [1], recombinant R-spondin 1 protein is used extensively in adult stem cell-derived organoid culture, intestinal organoid culture, hematopoietic stem cell specification and other iPSC and culture systems [1-5] .

Qk031 is an engineered form of R-spondin 1 with higher affinity for the LGR5 receptor and lower affinity for LGR4.  The aim is to specifically target Wnt-3a signalling in the LGR5+ stem cell population in intestinal organoids.

Qk006 recombinant human R-spondin 1 protein is a highly pure biologically active fragment comprising two cysteine-rich, furin-like domains – the essential domains for activity in stem cell culture. Our E. coli-produced recombinant R-spondin 1 protein is not glycosylated, which ensures reliable production of homogeneous protein and enhanced comparability between batches. The glycosylation of R-spondin 1 protein is not necessary for extracellular signalling and stability [6].

Background references

1. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011). doi.org/10.1038/nature10337
2. de Lau, W., Peng, W. C., Gros, P. & Clevers, H. The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 28, 305–316 (2014). doi: 10.1101/gad.235473.113
3. Genthe, J. R. & Clements, W. K. R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways. Development 144, 590–600 (2017). doi.org/10.1242/dev.139956
4. Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724–1743 (2016). doi.org/10.1038/nprot.2016.097
5. Broutier, L. et al. Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nat. Med. 23, 1424–1435 (2017). doi.org/10.1038/nm.4438
6. Chang, C.-F. et al. N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Binding Ability. doi:10.3390/ijms17060937

FAQ

What is the function of R-spondin 1?
R-spondin 1 regulates Wnt signaling and β-Catenin stabilization. Therefore, the functions of R-spondin 1 are diverse and are associated with cell proliferation and differentiation. It maintains the self-renewal capacity of stem cells and is involved in the embryonic development especially the development of the female reproductive system, gastrointestinal track, and neural tube.

What does the Wnt pathway do?
The Wnt signaling pathway is involved in in various biological processes. It is involved in the cell fate specification, embryonic development, stem cell maintenance, tissue regeneration, synaptic function, immune response, cancer progression, and cell migration.

What is the molecular weight of R-spondin 1?
The molecular weight is 13 kDa.

What does the RSPO1 gene do?
The RSPO1 gene provides instructions for producing the R-spondin 1 protein.

Where is the RSPO1 gene located?
The RSPO1 gene is located at 1q41.2.

How is recombinant R-spondin 1 used in cell culture?
It is a growth factor supplemented to cell culture media. It is used for the culture of induced pluripotent stem cells, embryonic stem cells, epithelial stem cells, and organoids such as intestinal and colon organoids.

How big is the R-spondin protein?
The size of the R-spondin proteins, including R-spondin 1 (RSPO1), can vary slightly depending on the specific isoform and the species being considered. In humans, RSPO1 is approximatively 263 amino acids in length with a molecular weight of 13-29 kDa.