Cloning, protein expression and purification

Expression constructs were prepared and verified using standard molecular biology techniques. Coding sequences for all proteins are of human origin. Mutant versions of ARIH1, UBE2L3, NEDD8 and ubiquitin were generated using Quikchange system (Agilent).

GST–TEV–RBX1 (residue 5 to C terminus), full-length CUL1, GST–TEV–UBA1, SKP1, His–TEV–β-TRCP2, GST–TEV–FBXL3 and cryptochrome 1 (CRY1, residues 1–532) were expressed in Trichoplusia ni High-Five insect cells, with CUL1 and RBX1, SKP1 and β-TRCP2, or SKP1, FBXL3 and CRY1 co-expressed as previously described^4,17. Proteins were purified by GST affinity chromatography followed by overnight TEV cleavage. Purification was completed by ion-exchange and size-exclusion chromatography. The neddylation components UBE2M and APPBP1–UBA3 were expressed in either Escherichia coli Rosetta (DE3) or BL21 Gold (DE3) cells as GST–thrombin fusion proteins. Fusion proteins were subjected to overnight protease cleavage and further purified by ion-exchange and size-exclusion chromatography. SKP1–FBXW7(∆D) (a monomeric version of FBXW7 comprising residue 263 to the C terminus)⁴⁷, SKP1–SKP2 (residues 101 to the C terminus), SKP1–SKP2 (full length) and the p27 N-terminal domain (residues 22–106) were expressed in E. coli Rosetta 2 (DE3) or BL21 Gold (DE3) and purified as described for neddylation components, but with overnight TEV cleavage after affinity purification. Neddylation of CUL1–RBX1 along with fluorescent labelling of ubiquitin were carried out as previously described⁵¹. UBE2M(Y130L) was used to modify CUL1–RBX1 with the I44A mutant of NEDD8⁵¹. ARIH1 (and mutant derivatives), UBE2D3, UBE2R2 and UBE2L3 (wild type, C86K and C17A/C137A) were expressed in E. coli Rosetta 2 (DE3) and purified by GST affinity chromatography. Overnight TEV cleavage was carried out on-column followed by size-exclusion chromatography of cleavage reactions. Expression and purification of cyclin A(170 to C terminus)–CDK2, NEDD8 and CKSHS1 (5–73) were performed as previously described⁴.