Purification and Characterization of Wnt Pathway Proteins

DVL2 DEP_417-511, DVL2 PDZ_265-361, FZD5 (GKTLESWRRFTS) and FZD8 peptide sequences (GKTLESWRALCTR) were tagged N-terminally with 6×His and Lip followed by a linker (ENLYFQS) encoding a cleavage site for tobacco etch virus (TEV) protease. Lip–Fz7 (encoding GKTLQSWRRFYH; Wong et al., 2003 (link)) was generated similarly, except that the TEV linker sequence was flanked by serine–glycine on both sides. Proteins were expressed in E. coli BL21-CodonPlus(DE3)-RIL cells (Stratagene) at 37°C, grown overnight at 24°C, and induced with isopropyl β-D-1-thiogalactopyranoside at OD 0.8. Lip–DEP, Lip–PDZ and Lip–FZDs were purified on NiNTA beads (Qiagen) essentially as described (Fiedler et al., 2011 (link)). SEC-MALS was performed in phosphate-buffered saline, using a GE Superdex S-200 10/300 analytical column, and analysed as described (Madrzak et al., 2015 (link)).