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Discovery and Characterization of a New Cell-Penetrating Protein
journal contribution
posted on 2013-12-20, 00:00 authored by Rudo L. Simeon, Ana Maria Chamoun, Thomas McMillin, Zhilei ChenWe describe a new cell-penetrating
protein, B1, capable of delivering
conjugated proteins and nucleic acids into mammalian cells. B1 is
a 244-amino-acid product of a single-base frameshift in the gene encoding
enhanced green fluorescent protein (eGFP). The molecule has a net
positive charge of 43 and a very high charge-to-mass ratio of 1.5.
eGFP-fused B1 potently penetrates both adherent and suspension cells
with >80% of cells taking up the protein when exposed to concentrations
as low as 1 μM. The protein was found to cluster in the paranuclear
region of TZM-bl cells. Most importantly, we show that B1 not only
facilitates cellular uptake but allows biomolecular cargo to reach
sites of biological relevance. For example, baby hamster kidney cells
underwent DNA recombination when exposed to B1-tagged Cre recombinase
at protein concentrations as low as 2.5 μM, indicating potent
nuclear delivery of functional protein cargos. Additionally, B1 delivers
noncovalently conjugated RNA and DNA across the cell membrane to cytosolic
and nuclear sites accessible to the cellular translation and transcription
machinery, as gauged by detection of encoded reporter functions, with
efficiency comparable to commercially available cationic lipid reagents.
B1 appears to utilize cell-surface glycans and multiple competing
endocytic pathways to enter and traffic through cells. These studies
provide both a new tool for intracellular delivery of biomolecules
and insights that could aid in the design of more effective cell penetrating
proteins.