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

## TL;DR

This paper provides a detailed and reproducible protocol for isolating and analyzing cell-penetrating peptides and supercharged proteins, using MeCP2 as a model to study their cellular uptake and function.

## Contribution

The study introduces a comprehensive and replicable workflow combining protein purification, buffer optimization, and live-cell imaging to analyze CPP-FPs and SPs.

## Key findings

- A workflow using DLS-guided buffer optimization yields stable CPP-FP/SP samples.
- Live-cell imaging distinguishes membrane-bound from internalized signals more accurately than plate-based methods.
- A CPP-like motif in MeCP2 is critical for its internalization, validated through multiple assays.

## Abstract

Cell-penetrating peptides (CPPs) and supercharged proteins
(SPs)
enable efficient intracellular delivery of macromolecules, with expanding
applications in basic research and in therapeutic development. 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. Live-cell fluorescence microscopy and imaging flow cytometry
enable discrimination between membrane-bound and internalized signal,
providing higher accuracy compared to plate-based readouts. MeCP2
sequence probing has revealed the presence of a CPP-like motif that
is critical to its internalization. Finally, validation assays clearly
demonstrated CPP-FP/SP activity. This protocol integrates advances
in protein biochemistry, structural analysis, and live-cell imaging
into a reproducible pipeline adaptable to a wide range of CPP- and
SP-based protein constructs and provides a practical framework for
downstream mechanistic and therapeutic interventions.