Superdex s 200 10 300 analytical column

Lip-DEP_416–511 was expressed in E. coli BL21-CodonPlus(DE3)-RIL cells (Stratagene) and purified essentially as described previously (Fiedler et al., 2011 (link)), and the tag was removed by tobacco etch virus (TEV) protease for NMR and crystallography (Supplemental Information). SEC-MALS was performed in PBS, using a GE Superdex S-200 10/300 analytical column, and analyzed as described previously (Madrzak et al., 2015 (link)). The NMR spectroscopy is described in the Supplemental Information.

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)).