Fig. 3

Protease specificity profiling. a Overview of phage substrate display. M13 bacteriophage libraries encode pIII fused to a FLAG-tag through a randomized protease substrate linker. These substrate phage are bound to anti-FLAG magnetic beads and treated with a protease to release phage that encode substrates that can be cleaved by the protease. The remaining intact substrate phage are eluted with excess FLAG peptide. The abundance of all substrate sequences within the cleaved and eluted samples is measured by high-throughput sequencing. b–e For all assayed proteases, phage substrate display was separately performed on seven libraries, each with a different single randomized position within the ENLYFQS motif. The results are displayed as sequence logos, with letter height proportional to enrichment in the cleaved versus eluted sample. Letters placed above the x-axis indicate protease acceptance and letters beneath the axis indicate rejection. b Wild-type TEV protease exhibits strong enrichment for the consensus motif EXLYFQS. c Evolved TEV L2F has broadened specificity at P6 and shifted specificity at P3, P1, and P1ʹ in accordance with the HPLVGHM target substrate. d Mutations I138T, N171D, and N176T are sufficient to broaden P6 specificity. e Mutations T146S, D148P, S153N, S170A, and N177M shift specificity at both P1 and P3