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Product description:
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Plasmid is available in 10 ug aliquots. Full sequence available on request. R115L is a diseaseÄËĂÂĂÂassociated mutation 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 (TALONÄÂÄšËClontech), 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. Recombinant protein can be stored in 10mM acetate buffer, pH 4.0, 0.5 M NaCl, 0.5% [w/v]CHAPS or in 10 mM HEPES, pH 7.5, 0.7 M NaCl, 0.5% [w/v] CHAPS. > Norrin (residues 25-133) R107E/R109E/R115L KTDSSFIMDSDPRRCMRHHYVDSISHPLYKCSSKMVLLARCEGHCSQASRSEPLVSFSTVLKQPFRSSCHCCRPQTSKLKALELECSGGMLLTATYRYILSCHCEECNS
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Additional notes:
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Norrin (Norrie Disease Protein) is a cystine-knot like growth factor that can activate Wnt signalling by binding to Frizzled and another receptor protein called Lrp5/6. This group or ‘complex’ also includes molecules called glycosaminoglycans. The R107E/R109E/R115L mutant loses signalling activity and binding ability to HSPGs co�receptor, but retains the interactions with Frizzled 4 receptor and co�receptors of low density lipoprotein related protein 5/6 (Lrp5/6). 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.
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