g peptides on the activity of cullinRING ligase in the cell. APPBP1-Uba3 between the NheI and NotI restrictions sites was replaced with the gene of the peptidyl carrier protein domain of GrsA with an N-terminal 66His tag to generate the plasmid pGEX-PCP-APPBP1-Uba3. This plasmid was used for the coexpression of the PCP-APPBP1 fusion and Uba3 to assemble PCP-NAE. For the expression of HA-Nedd8 with an N-terminal HA tag, the Nedd8 gene was amplified by polymerase chain reaction from pGEX-NEDD8 and cloned into pET28a between the NheI and NotI restriction sites. The pET-HA-Nedd8 plasmid expressed HA-Nedd8 fusion with an N-terminal 66His tag. Protein Expression and Purification For the expression of proteins with a 66His tag from the pET or pGEX vectors, the plasmids were transformed into the BL21pLysS chemical competent cells and plated on LB-agar plates with appropriate antibiotics. Protein expression and purification 19053768 followed the protocol provided by the vendors of the pET expression system and the Ni-NTA agarose resin. Typically the cells were grown in 1 L Lysogeny Broth supplemented with 100 mg/mL ampicillin to 
an OD around 0.60.8 at 37uC. The LB culture was then induced by adding IPTG to a final concentration of 1 mM and incubating with continuous shaking at 15uC overnight. Cells were subsequently collected by centrifugation at 5,000 rpm for 10 min, resuspended in lysis buffer and lysed with BQ-123 site French press. The resulting crude suspension was centrifuged at 12,000 rpm to remove the pelleted cell debris from protein lysate. The lysate supernatant was mixed with 1 mL of Ni-NTA agarose and rocked gently at 4uC for 2 hours. The slurry was next transferred to a gravity column, washed once with 15 mL lysis buffer, twice with 15 mL wash buffer and eluted with 5 mL elution buffer. Eluted protein was dialyzed overnight at 4uC in a 1 L buffer, followed by a second dialysis the next day with the same buffer for 3 hours. All purified proteins were assayed by electrophoresis on a 4 15% SDS Tris polyacrylamide gel for the verification of their sizes and purity, and eventually stored in aliquots at 280uC. Ubc12 and the cullin3-Rbx1 complex was expressed and purified as previously reported. Biotin Conjugation to PCP-NAE Fusion Biotin labeling of 17372040 PCP-NAE catalyzed by Sfp phosphopantetheinyl transferase followed a reported protocol. 100 mL labeling reaction was set up containing 5 mM PCP-NAE, 2 mM biotin-CoA, 0.3 mM Sfp in a reaction buffer. The reaction was allowed to proceed for 1 hour at 30uC, and then mixed with 100 mL 3% BSA. 100 mL of the reaction mixture was distributed to a 96-well plate coated with streptavidin and allowed to bind to the plate for 1 hour at room temperature. The plate was then washed three times with TBS buffer to remove unbound enzymes before the phage selection reaction. Materials and Methods Molecular Cloning Enzyme-linked Immunosorbent Assay The transfer of Nedd8 to biotin labeled PCP-NAE bound to the streptavidin plate was analyzed by ELISA. After the labeling reaction, PCP-NAE attached with biotin was bound to a streptavidin plate and the plate was washed with TBS. 5 mM Nedd8 protein with an N-terminal HA tag was added to the Nedd8-Like Ubiquitin Variants streptavidin plate in the presence of 5 mM ATP in TBS. Control reactions were also set up excluding ATP or using a streptavidin plate without the coating of PCP-NAE. After a 1 hour incubation, the plate was washed and the Nedd8 protein bound to the plate was detected by bin
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