Fig 1: Protease-mediated attenuation of IL-17 secretion in mouse splenocytes. The activity of IL-23 in vivo is mediated by stabilization of a T-helper cell lineage (Th17) that secretes IL-17, leading to downstream pro-inflammatory signals. This pathway can be assayed within a culture of mouse mononuclear splenocytes, by measuring the amount of IL-17 secretion into the cell culture media using an ELISA. As a positive control, anti-IL-23 antibodies in a super-stoichiometric ratio prevent IL-17 secretion. Preincubation of IL-23 and evolved TEV L2F attenuates IL-17 secretion, demonstrating that cleavage of the HPLVGHM target site inactivates immune signaling capabilities of IL-23. Values represent the mean and error bars represent the standard deviation of three technical replicates
Fig 2: PACE evolutionary trajectories. Across the eight stages of PACE along three diverging trajectories (shown in purple, blue, and orange), each arrow represents a PACE experiment with the corresponding substrate peptide and selection stringency parameters listed beneath the arrow. Increased selection stringency annotations are: Q649S (a T7 RNAP mutant with decreased transcriptional activity), proA (lower expression of substrate PA-RNAP), and IL-23 (38–66) (native IL-23 sequence in place of GGS linker). Numbers above the arrows denote TEV protease residues that were targeted in site-saturation mutagenesis libraries used to initiate that PACE experiment. In the first PACE experiment, wild-type TEV protease was mutagenized at the positions shown. All other libraries were generated using the protease genes emerging from the previous PACE stage as the PCR template. For PACE stages with no targeted mutagenesis, lagoons were inoculated with an aliquot of the phage population from the preceding experiment
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