Abstract
Protein therapy holds great promise for treating a variety of diseases. To act on intracellular targets, therapeutic proteins must cross the plasma membrane. This has previously been achieved by covalent attachment to a variety of cell-penetrating peptides (CPPs). However, there is limited information on the relative performance of CPPs in delivering proteins to cells, specifically the cytosol and other intracellular locations. Here we use green fluorescent protein (GFP) as a model cargo to compare delivery capacity of five CPP sequences (Penetratin, R8, TAT, Transportan, Xentry) and cyclic derivatives in different human cell lines (HeLa, HEK, 10T1/2, HepG2) representing different tissues. Confocal microscopy analysis indicates that most fusion proteins when incubated with cells at 10 µM localise to endosomes. Quantification of cellular uptake by flow cytometry reveals that uptake depends on both cell type (10T1/2 > HepG2 > HeLa > HEK), and CPP sequence (Transportan > R8 > Penetratin≈TAT > Xentry). CPP sequence cyclisation or addition of a HA-sequence increased cellular uptake, but fluorescence was still contained in vesicles with no evidence of endosomal escape. Our results provide a guide to select CPP for endosomal/lysosomal delivery and a basis for developing more efficient CPPs in the future.
Highlights
The delivery of functional proteins to cells offers potential for therapeutic intervention
We compare the ability of five Cell-penetrating peptides (CPPs) (Penetratin, R8, TAT, Transportan, Xentry) and their derivatives under identical experimental conditions to promote the cellular uptake of green fluorescent protein (GFP) in four cell lines of different tissue origins (HeLa – human cervical epithelial, HEK – human kidney epithelial, 10T1/2 – mouse embryonic, HepG2 – human liver epithelial)
It is known that addition of a CPP sequence can have a negative influence on recombinant protein yield[10], we swapped the position of CPP and His tag in the pBAD plasmid (Fig. S1a) and attempted to produce superfolder GFP (sfGFP)-R10 (Fig. S1b) in TOP10 cells
Summary
The delivery of functional proteins to cells offers potential for therapeutic intervention. Unlike conventional small-molecule drugs, most proteins are large and hydrophilic Such characteristics mean they do not pass directly through the plasma membrane which restricts their use as therapeutics. Cell-penetrating peptides (CPPs) have shown potential for the delivery of a wide range of molecules, including large active proteins to enter cells via endocytosis[4,5,6]. A thorough comparative investigation into the relative potential of different CPPs for intracellular delivery of protein cargos is missing[12,13,14]. The results presented here indicate a very large variation in efficiency of cellular uptake as a function of CPP sequence, cyclisation and cell line. The analysis provides a helpful guide for the choice of CPP for cargo delivery into endosomes and lysosomes, and a basis for developing more efficient CPPs in future
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