Recombinant R-spondin 3 protein (Qk032)2020-02-29T20:47:55+00:00

Recombinant human R-spondin 3 (Qk032)

R-Spondin-3 (RSPO3) protein belongs to the R-spondin family comprising four structurally-related, secreted ligands (RSPO 1-4) (1). All contain furin-like and thrombospondin structural domains. R-spondins are activators of the canonical Wnt signaling pathway. Receptors include the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-6 (2). In the colon, R-spondin 3 promotes stem cell recovery and epithelial repair via induction of Wnt.

Qkine R-spondin-3 protein is a highly defined, animal-free pure biologically active protein fragment comprising the two cysteine-rich, furin-like domains, which are necessary and sufficient for Wnt signal potentiation and are the essential domains for activity in organoid culture.

An R-spondin such as R-spondin-3 is an essential component of Wnt 3a, R-spondin-3 (or 1,2), Noggin (WRN) media used widely in propagation of intestinal organoids (2).  There is a drive towards improved reproducibility in stem cell and organoid culture.  Conditioned media (spent media from cells that secrete growth factors such as Wnt and R-spondin, along with several other factors and waste products) is both challenging to manufacture consistently and conveniently, and a source of variability between labs (3).

We produce R-spondin-3 in E.coli, so the protein is not glycosylated and is produced in animal-derived component free laboratory making it ideal for formulation of defined media. The glycosylation of R-spondin-3 is unnecessary for extracellular signalling and stability. Our methods allow for optimised reliable production of homogeneous protein and enhanced comparability between batches. We provide bulk pricing and batch stock reservation for this protein.

Summary: bioactive domain of human R-spondin 3 (Uniprot: Q9BXY4)

Form: protein is provided lyophilized from a fully volatile solution without carrier protein.  Animal-derived component free. 

Molecular mass: ~17 kDa

Please follow the handling guidance for lyophilised cytokines below to minimise loss of protein due to precipitation or adsorption to plastic.  We advise storing the recombinant protein at very low pH to before dilution in cell culture media or final working solutions. Low pH will also assist in maintaining the correct disulphide structure of the protein by minimising disulphide bond exchange reactions.

  • Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilised cytokines in 10 mM HCl (1:1000 dilution of concentrated HCl) while keeping the protein concentration at 50 µg/ml or above, in order to avoid loss by adsorption to plasticware.
  • To ensure you recover all of the protein, let the sample sit for a few minutes with the solubilisation buffer at room temperature and pipette gently up and down (avoid foaming).
  • Rinse the tube with some more 10 mM HCl and pool with the rest.
  • The protein is tolerant of some freeze and thaw cycles, but as always with proteins, it is better to aliquot and stored frozen.
  • Our protein are supplied carrier-protein free.  If compatible with your work, add carrier protein of your choice such as BSA, HSA or gelatin to further minimise loss by adsorption.
  • Store in -80°C for long term storage. -20°C for short-term.

1. de Lau, W. B. M., Snel, B. & Clevers, H. C. The R-spondin protein family. Genome biology 13, 242 (2012).
2. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
3. VanDussen, K. L., Sonnek, N. M. & Stappenbeck, T. S. L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams. Stem cell research 37, 101430 (2019).

R-spondin-3, RSPO3, RSPO-3, Protein with TSP type-1 repeat, PWTSR, Roof plate-specific spondin-3, Thrombospondin type-1 domain-containing protein 2, THSD2

Result: R-spondin 3 bioactive domain migrates at 17 kDa in non-reducing (-βME) conditions and upon reduction (+βME).

Recombinant R-spondin-3 protein purity in SDS-PAGE

Purified recombinant protein (7 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.

Result: RSPO 3 enhances Wnt signalling in the TOPflash reporter assay with EC50 = 0.49 nM

Bioactivity of R-spondin 3 in Wnt TOP FLASH assay

Bioactivity using a Wnt reporter assay in HEK293T cells.   HEK293T cells transfected with Wnt-responsive firefly luciferase reporter TOPflash are treated with increasing concentrations of Qk032 R-spondin 3 #010 in the presence of Wnt-conditioned media (1:8 dilution), in triplicate. The following day, luciferase activity is measured by luminescence.

Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.

Protein purity and structural homogeneity is analysed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analysed in ACE C4 4.6 x 250 mm column using eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid . Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient. Example data from Qk001 batch #011

Protein purity and structural homogeneity is analysed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analysed in ACE C4 4.6 x 250 mm column using eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid . Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient.  Example data from Qk032 batch #010

Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.

Recovered concentration: 1.05 mg/ml or Recovery: 105%

Check full recovery from vial of R-spondin 3 protein using UV spectra

The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. This was diluted 1:5 in 6 M guanidine hydrochloride, 20 mM sodium phosphate pH 7.4 and the UV spectrum from 340-220 nm read. The concentration was calculated using the extinction coefficient at 280 nm.  Example data from batch #010.

Result: Endotoxin level <0.005 EU/ug protein (below level of detection)

Stem cell cultures are sensitive to endotoxins1, which can be present in media, serum and as a contaminant on plasticware.  We optimize our protein production processes to ensure the lowest possible levels of endotoxin contamination.    Our endotoxin pass criteria are set at the industry leading <0.1 EU per ug protein and we aim for <0.01 EU per ug protein.  Endotoxin levels in our proteins are determined by an external expert microbiological testing services provider.

1. A biological study establishing the endotoxin limit for in vitro proliferation of human mesenchymal stem cells (2017). Yusuke Nomura, Chie Fukui, Yuki Morishita, Yuji Haishima. Regenerative Therapy, 7, 45-51.

All our proteins are produced in our Cambridge, UK, labs.  We provide detailed quality data for each batch because we believe reliable, high quality cytokines and growth factors are critical for successful stem cell and organoid culture.

When we test our proteins, we choose a vial at random and reconstitute as recommended to ensure we are testing as close to the protein you will receive as possible.  Biochemical identity and purity is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography.  Bioactivity is determined using an appropriate cell-based assay.  As stem cells are sensitive to endotoxin levels, we use a high resolution test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein).  We also check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so when you use the proteins you can rely on your calculated dilution.

Please contact us with questions any time by email support@qkine.com or phone +44 (0) 1223 491486 / US toll free 1-866 877 2185.

Order online by clicking below or email orders@qkine.com

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