Human R-spondin 1 LR5 Qk031 protein bioactivity lot #104286

Human R-spondin 1 LR5 Qk031 protein purity lot #104286

Recombinant human R-spondin 1 LR5 protein (Qk031)

Name: Recombinant human R-spondin 1 LR5 protein (Qk031)
SKU: Qk031
Availability: InStock

Recombinant human r-spondin 1 LR5 protein is engineered to act as a high affinity ligand for the LGR5 receptor. In epithelial tissues LGR5 marks the stem cell population. This engineered protein, R-spondin 1 LR5, activates wnt signalling only in the LGR5+ stem cell population. R-spondin1 LR5 has been tested in intestinal organoid culture and supports organoid survival and growth. As LGR5 specifically marks stem cells and is not found on transit amplifying cells, the lower crypt multiplicity seen in organoid cultures with R-spondin 1 LR5 (in comparison to wild-type R-spondin 1, Qk006) supports the notion that this engineered form is acting specifically on stem cells. This specialized form of R-spondin 1 was developed in Marc de la Roche’s lab (University of Cambridge).

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1mg will be despatched as 2 x 500µg

Description

Summary

High purity recombinant human r-spondin 1 LR5 protein. Binds with higher affinity to LGR5 receptor
>98%, by SDS-PAGE quantitative densitometry
13 kDa
Expressed in E. coli
Animal-free (AOF) and carrier protein-free.
Manufactured in our Cambridge, UK laboratories
Lyophilized from acetonitrile, TFA
Resuspend in 10mM HCl at >100 µg/ml, prepare single use aliquots, add carrier protein if desired and store frozen at -20°C or -80°C

Handling and Storage FAQ

Featured Applications

Intestinal organoid culture

Alternative protein names

Roof plate-specific spondin 1, Rspo1, R-spondin 1, Cristin 3

Background references

1. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).

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).
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).
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).
5. Broutier, L. et al. Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nat. Med. 23, 1424–1435 (2017).
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.

Alternative

Recombinant human R-spondin 1 protein

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 = 47.2 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.

Further quality assays

Mass spectrometry: single species with expected mass
Analytical reversed-phase: single sharp peak
Endotoxin: <0.005 EU/μg protein (below level of detection)
Recovery from stock vial: >95%

Request CoA/animal-free certificate

Download MSDS

We are a company founded and run by scientists to provide a service and support innovation in stem cell biology and regenerative medicine. All our products are exceptionally high purity, with complete characterisation and bioactivity analysis on every lot.

R-spondin 1 LR5 specifically activates Wnt signalling in lgr5+ stem cell population

Mouse intestinal organoid morphology is healthy in cultured maintained in R-spondin 1 LR5

Mouse intestinal organoid culture morphology

All organoid data from the laboratory of Marc de la Roche and reproduced with permission (© Marc de la Roche, 2020).

Intestinal organoid survival is maintained in R-spondin 1 LR5 cultures

Lower crypt multiplicity is observed suggesting targeting of LGR5+ stem cell population

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.

Recombinant human r-spondin 1 LR5 protein support organoid survival and growth in intestinal organoid culture. As LGR5 specifically marks stem cells and is not found on transit amplifying cells, lower crypt multiplicity is seen in organoid cultures with R-spondin1 LR5 (in comparison with WT R-spondin1), which supports the notion that this engineered form is acting specifically on stem cells.

This is part of our pioneering proteins range where the full range of activities and applications of this novel protein have not been determined.

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) 4-6¹ , 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.

Our products are for research use only and not for diagnostic or therapeutic use. Products are not for resale.

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