Extended Data Fig. 9: Generation and validation of a synthetic human Fab phage library.
From: A conformational switch controlling the toxicity of the prion protein
Extended Data Figure 9. (a) A synthetic human Fab phage library was used for panning. For each panning round, the targeted antigens are reported with the respective concentration. Full-length recombinant murine PrP23–231 (rmPrP23–231; light blue boxes) was used as a target for the first and the second round of phage panning. At the third and fourth round, phages were depleted of the binders to rmPrP2cys and selected for binding to either rmPrP90–231 or rmPrP121–231 (recombinant murine PrP fragments lacking the N-terminal flexible tail; light red boxes) or to recombinant human PrP23–230-AviTagTM (rhPrP23–230-AviTagTM, purple boxes). In rmPrP90–231 or rmPrP121–231 panning, Fab-displayed Fab were depleted of binders to rmPrP2cys coated on plates. In rhPrP23–230-AviTagTM panning, depletion of binders to rmPrP2cys in solution was achieved by capturing Fabs binding to rhPrP23–230-AviTagTM on neutravidin coated wells. Polyclonal DNA preparation from the selected phages at the third round (rmPrP90–231) and fourth round (rmPrP121–231 and rhPrP23–230-AviTagTM) was used for transformation in bacteria and the screening of single clones by ELISA. (b) ELISA (OD at 450 nm) comparing the reactivity of phage-derived anti-PrP Fabs to full-length rmPrP23–231, FT fragment rmPrP23–110 and GD fragments rmPrP90–231 and rmPrP121–231. Anti-PrP Fab100 and Fab53 bind within the FT of PrP - the octapeptide repeat region (OR, amino acid 51–90) and the charged cluster 2 (CC2, amino acid 93–100), respectively. FabA10, FabD9, FabE6 and FabE2 bind within the GD. Error bars = standard error of the mean. One datapoint corresponds to a technical replicate in a multi-well plate.
