Human Norrin Protein

Human Norrin Protein

Recombinant mature form (amino acids 25-133) of human norrin expressed in HEK-293T cells and purified utilizing affinity and size exclusion chromatography.

Highlights:

  • Highly Pure - >99% (SDS-PAGE)
  • Biologically Active - Shown to active the canonical Wnt/β-catenin pathway in luciferase reporter assay

Norrin (Norrie Disease Protein) is a cystine-knot like growth factor that can activate the canonical Wnt signaling pathway through FZD4 and LRP5/6 coreceptor. This group or ‘complex’ also includes molecules called glycosaminoglycans. Norrin plays a central role in retinal vascularization by acting as a ligand for FZD4 that signals via stabilizing beta-catenin (CTNNB1) and activating LEF/TCF-mediated transcriptional programs. The protein also acts in concert with TSPAN12 to activate FZD4 independently of the Wnt-dependent activation of FZD4, suggesting the existence of a Wnt-independent signaling that also promote accumulation the beta-catenin (CTNNB1). Norrin may be involved in a pathway that regulates neural cell differentiation and proliferation. Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. In humans, mutations in the gene that encodes Norrin can cause a disease in which blood vessels in the eye fail to form correctly, which can result in blindness. However, it is not clear how Norrin activates Wnt signalling.

From a laboratory at Cancer Research Technology.

Catalog Number Product DataSheet Size AVAILABILITY Price Qty
EW0007
Human Norrin Protein
25ug Currently unavailable
Regular Price:$355.00
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Specifications

Product Type: Protein
Name: Norrin (Norrie disease protein, X-linked exudative viteoretinopathy 2 protein)
Accession ID: Q00604
Source: Recombinant expression in HEK-293T Cells
Molecular Weight: ~15 kDa (full length monomer)
Amino Acid Sequence: KTDSSFIMDSDPRRCMRHHYVDSISHPLYKCSSKMVLLARCEGHCSQASRSEPLVSFSTVLKQPFRSSCHCCRPQTSKLKALRLRCSGGMRLTATYRYILSCHCEECNS
Fusion Tag(s): C-terminal TETSQVAPA sequence derived from bovine rhodopsin (Rho-1D4) which is recognized by the Rho-1D4 monoclonal antibody
Purity: Affinity and SEC purified
Buffer: 10mM acetate buffer, pH 4.0, 0.5 M NaCl, 0.5% [w/v] CHAPS. (Can also be kept in 10 mM HEPES, pH 7.5, 0.7 M NaCl, 0.5% [w/v] CHAPS)
Concentration: 25ug
Storage: -80 °C; Multiple freeze/thaw cycles are not recommended
Shipped: Dry ice

Data

Purification and Biological Activity

NorrinData

(Left) SEC elution profile and SDS-PAGE under reducing conditions with fraction analysed marked by red lines. (Right) Purified recombinant untagged Norrin actives the canonical Wnt/?-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).

Adapted from: Chang TH, et al. Elife. 2015 Jul 9;4. doi: 10.7554/eLife.06554.

Provider
From a laboratory at Cancer Research Technology.
Comments
Norrin recombinant protein expression and purification protocol: Norrin was expressed in HEK293T cells in the presence of 4 mM valproic acid, a histone deacetylase inhibitor (Backliwal et al., 2008), used to increase the expression level of secreted protein. The detailed expression protocol was described in our publication (Chang et al., 2015). Norrin conditioned media (500 ml) were dialyzed against 5L of PBS buffer plus 0.4 M NaCl for 24 hrs. The dialyzed media were adjusted to 20 mM Tris, pH 8.0 and 2.5 mM Imidazole, pH 7.5. Recombinant protein was further purified from the adjusted media by IMAC (TALON®Clontech), washed with 25 mM Tris, pH7.5, 0. 5 M NaCl, 0.02 M Imidazole, 10 % [w/v] Glycerol, and eluted in 25 mM Tris, pH7.5, 0.15 M NaCl, 0.5 M Imidazole. The purified sample was added CHAPS to 1% [w/v] and dialyzed against 25 mM Tris, pH 7.5, 1M NaCl, 10% [w/v] Glycerol, before treating with His-tagged HRV-3C protease to remove the SUMOtagged fusion protein. The untagged sample was further isolated by IMAC (collection of flow-through and wash with 25 mM Tris, pH7.5, 1 M NaCl, 0.01 M Imidazole, 1% [w/v] CHAPS) and purified by SEC (SEC, Superdex 200 10/300 GL High Performance, GE Healthcare Life Sciences) in 10 mM HEPES, pH 7.5, 0.7 M NaCl, 0.5% [w/v] CHAPS or 10 mM acetate buffer, pH 4.0, 0.5 M NaCl, 0.5% [w/v] CHAPS.

References
  1. Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan. Elife. 2015 Jul 9;4. doi: 10.7554/eLife.06554.

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