Journal: Scientific Reports
Article Title: A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery
Figure Lengend Snippet: 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.
Article Snippet: Specifically, the Phylomer CPP retained EGFR-dependent specificity when combined with a targeting Affibody, and also largely maintained potency after PASylation .
Techniques: Conditioned Place Preference, Selection, Binding Assay, Activity Assay, Cell Surface Receptor Assay, Expressing, Sequencing, Labeling, Magnetic Beads, Amplification, Clone Assay