Figure 1
CPP screening and selection process. (a) Selection begins with a T7 phage library displaying a fusion of a cargo (in this example, an EGFR receptor binding domain, EBD), an Avitag and Phylomer peptides; (b) Avitagged-Phylomer sequences with potential for CPP activity are internalized into cells; this uptake can also be facilitated by binding to specific cell types via a cell surface receptor (CSR) and uptake into endosomes by receptor-mediated endocytosis; (c) peptides with capacity for cytosolic delivery allow the phage to enter the cytoplasm; (d) selection is performed in mammalian cells expressing the bacterial biotin ligase BirA in the cytoplasm, which ligates free biotin to the lysine residue of the phage-displayed Avitag sequence; this step produces selectively labeled T7 phage that have internalized into the cytoplasm by virtue of the CPP; (e) sodium pyrophosphate (PPi), a specific inhibitor of BirA, is added to the cells to terminate the biotinylation reaction; (f) cells are lysed and streptavidin-coated magnetic beads (SAV) are added to the lysate to selectively capture and concentrate biotinylated T7 phage. Enriched phage are then amplified in E. coli and subjected to further rounds of selection. Identification of specific CPPs is achieved by deep sequencing of early selection rounds or by Sanger-sequencing of individual phage clones after 3–4 rounds of selection.
