Purification of C-Raf and Related Proteins

WT human C-Raf and C-Raf-carboxyl terminus were expressed and purified as previously described (36 (link)). Briefly, full-length human C-Raf or C-Raf-carboxyl terminus with an N-terminal GST tag were expressed in sf9 cells. The proteins were extracted and purified on glutathione sepharose beads (GoldBio). Then the GST tag was cleaved off by thrombin protease. Finally, C-Raf was further purified by size-exclusion chromatography (SEC). GST-C-Raf-amino terminus, GST-C-Raf-CR1, GST-C-Raf-RBD, and GST-C-Raf-CRD were expressed in Escherichia coli BL21 (DE3) cells and purified on glutathione sepharose beads (GoldBio) followed by further purification using SEC. Expression and purification of Fab30 (37 (link)), H-Ras (38 (link)), and WT βarr1 and its variants (39 (link)) have been described previously. Expression and purification of FLAG-M2R with C-terminal sortase ligation consensus sequence (LPETGGH) followed by a 6× His-tag was performed as previously described (34 (link)).