Fig 2: Vector maps of pPb EF1α-EpCAM-EGFP (A) and pPb EF1α-NP65-EGFP (B). The expression of both fusion proteins EpCAM-EGFP and NP65-EGFP was under control of EF1α promoter. Both expression vectors encoded for puromycin resistance gene. DNA sequences which should be integrated into the host genome were flanked by inverted terminal repeat (ITR, red) sequences. They were recognized by piggybac transposase (encoded on a separate helper plasmid), cutted out and integrated into TTAA chromosomal sites of host genome. For vector propagation in E.coli strains, both expression vectors possessed bacterial origin of replication (ori, yellow)

Fig 3: In silico prediction for the localization of domains of the fusion membrane proteins EpCAM-EGFP (A) and NP65-EGFP (B) using the online tool TMHMM-2.0 (DTU Health Tech). The probability for the localization of domain (intracellular, transmembrane or extracellular) was calculated at each amino acid position and presented as a graph

Fig 4: Cell-based selection round with the stable cell line HEK293-NP65-EGFP. 5 × 1014 phage particles from the human naïve scFv phage library were pre-incubated with 1 × 106 wildtype HEK293 cells. Unbound phage particles were added to 1 × 106 cells expressing the extracellular domain of NP-65 as fusion protein with EGFP (intracellular domain). Both approaches were incubated with an anti-phage antibody conjugated with DyLight® 650. Fluorescence intensities for those cell populations were presented as histograms in DyLight® 650 channel. The sorting gate was set up according to the negative control. Moreover, sort populations were presented in the EGFP channel for cross checking the expression of NP65 extracellular domain