VECTORS
Contributor Information
- Name Tao-Hsin Chang ; E. Yvonne Jones
- Institute University of Oxford
Tool Details
- Tool name: Norrin Mutant [L61N/A63S] Vector
- Tool type: Vector
- Tool sub-type: pHLIgK-STR-8H-SUMO-1D4
- Description: Plasmid is available in 10 ug aliquots. Full sequence available on request. 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 (TALONĂĹ˝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. 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) L61N/A63S KTDSSFIMDSDPRRCMRHHYVDSISHPLYKCSSKMVNLSRCEGHCSQASRSEPLVSFSTVLKQPFRSSCHCCRPQTSKLKALRLRCSGGMRLTATYRYILSCHCEECNS
- Research area: Neurobiology; Stem Cell Biology
- For Research Use Only
Target Details
- Target: Human Norrin (Norrie Disease Protein, NDP)
Application Details
Handling
Documentation
- Available on request
References
- • Chang et al. 2015. Elife. 4:. PMID: 26158506.
- • Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.
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