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. 2018 Jan 3;4(1):e1701393. doi: 10.1126/sciadv.1701393

Fig. 1. Phage selection for the orthogonal xUbcH5b-xU-box pair for E4B.

Fig. 1

(A) wt UB labeled with biotin (biotin–wt UB) was activated by xUba1(UFD) and loaded on xUbcH5b. The U-box library of E4B was displayed on the surface of M13 phage. Phage displaying U-box mutants reactive with xUbcH5b were conjugated with biotin-UB and selected by binding to streptavidin. ATP, adenosine 5′-triphosphate; AMP, adenosine 5′-monophosphate. (B) Sequence alignment of U-box mutants of E4B from phage selection. Sites of randomized residues in the loop1 of the E4B U-box are marked by red asterisks. After five rounds of selection, 30 phage clones were sequenced; the alignment of the sequences showed that the phage library was converged to KB2 and other highly homologous clones.