Figure 2.

Phage selection of the Nedd8 library based on its reactivity with NAE. A) The phage selection scheme. The Nedd8 protein was displayed on the surface of M13 phage with the pJF3H phagemid that expressed Nedd8 with an N-terminal Fos peptide so that Nedd8 could be anchored on the phage surface by high affinity binding of the Jun-Fos peptides. The pJF3H phagemid gave rise to monovalent display of Nedd8 variants on phage particles. C-terminal residues of Nedd8 covering the sequence ⁷¹LALRG⁷⁵ were randomized (“X” in black). Phage selection specifically enriched C-terminal sequences of Nedd8 variants that can be recognized by NAE for activation (“X” in red). B) Titer of the eluted phage from each round of Nedd8 library selection with NAE. In the selection reaction, both NAE and ATP were provided for the formation of Nedd8~NAE thioester conjugates. In the control reactions, either ATP or NAE was excluded from the reaction mixture. The titer numbers of the phage were plotted on a logarithmic scale (cfu, colony forming unit).