Ubiquitin purification, substrate ubiquitination, and preparation of unanchored ubiquitin chains

E. Coli Bl21*(DE3) cells were transformed with a pET28a plasmid coding for yeast ubiquitin, grown in terrific broth at 37°C until OD₆₀₀ = 0.6 to 0.9; induced with 1 mM IPTG overnight at 18°C; pelleted; resuspended in lysis buffer with lysozyme, benzonase, and protease inhibitors; and lysed by sonication. The lysate was clarified by centrifugation, and its pH was adjusted to 4.5 using acetic acid. Protein precipitate was removed by centrifugation, and the supernatant was dialyzed against 50 mM Na-acetate (pH 4.5) overnight at 4°C. Ubiquitin was purified by cation-exchange chromatography with a 5-ml HiTrap SP FF column (Cytiva) and a gradient of 0 to 0.5 M NaCl in 50 mM Na-acetate (pH 4.5), followed by size exclusion chromatography using a Superdex 75 16/60 column equilibrated with GF buffer. Enzymatic addition of ubiquitin chains to substrates containing PPPYX motifs and a single lysine residue was performed as previously described (4), incubating 10 μM substrate for 3 hours at 25°C in GF buffer with 10 mM ATP, 400 μM ubiquitin, 2.5 μM mouse E1, 2.5 μM Ubc1, and 25 μM Rsp5. K48-Ub₄ and K63-Ub₄ were synthesized and purified as previously described (58). K48-linked chains were synthesized by incubating 1 mM ubiquitin with 1 μM mE1, 5 μM Cdc34, and 10 mM ATP in GF buffer overnight at 37°C. K63-linked chains were synthesized by incubating 1 mM ubiquitin with 1 μM mE1, 2.5 μM Ube1a/UbcH13, and 10 mM ATP in GF buffer overnight at 37°C. Tetrameric ubiquitin chains were separated from the other chain lengths using a Resource S cation-exchange column (Cytiva) with a 0 to 600 mM NaCl gradient and a Superdex 75 increase 10/300 (Cytiva) in GF buffer and were verified by gel electrophoresis. Linear tetra-ubiquitin chains were prepared as previously described (4): A linear hexa-His SUMO (small ubiquitin-like modifier)–tetra-ubiquitin fusion was affinity purified using Ni-NTA, cleaved with the protease SENP2, and then purified over a Superdex 75 16/60 (Cytiva) in GF buffer.