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Figure 1. Expression and purification of biologically active recombinant Norrin.(A) Schematic diagrams of the expression constructs including Norrin (a signal peptide, SP, followed by Norrin and Rho-1D4 tag at C-terminus) and SUMO-Norrin (a SP followed by a Strep-tag II, an octahistidine, SUMO, HRV 3C protease cleavage site, Norrin, and Rho-1D4 tag at C-terminus). (B and C) Conditioned media from transfected HEK293T cells were immunoblotted (IB) with the anti-Rho-1D4 antibody. (B) SUMO fusion improves Norrin secreted expression. (C) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid. (D) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines. (E) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).DOI: http://dx.doi.org/10.7554/eLife.06554.003 Image published in: Chang TH et al. (2015) © 2015, Chang et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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