Cell-Penetrating Peptides and Supercharged Proteins: A Comprehensive Protocol from Isolation to Cellular Uptake

Despite their potential, reproducible workflows for isolation, biochemical characterization, and quantitative uptake analysis remain limited. Here, we present a comprehensive and replicable protocol for the isolation, characterization, and cellular uptake analysis of CPP-fusion proteins (CPP-FPs) and SPs using methyl-CpG-binding protein 2 (MeCP2) constructs as a proof-of-principle model.

This workflow combines native protein purification with dynamic light scattering (DLS)-based buffer optimization. Cellular uptake is then assessed and quantified under live-cell conditions using high-content imaging and imaging flow cytometry, with additional assays to probe endocytic trafficking routes, identify CPP-like motifs in SPs, and validate transducing CPP-FP/SP functionality. The protein isolation and DLS-guided buffer screen yield samples with long-term stability.