Arginine Grafting to Endow Cell Permeability

Fuchs, Stephen M.; Raines, Ronald T.

doi:10.1021/cb600429k

Cited by 73 publications

(73 citation statements)

References 31 publications

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“…When the replacement residues are all positively or all negatively charged, the resulting ''supercharged'' proteins can retain their activity while gaining unusual properties such as robust resistance to aggregation and the ability to bind oppositely charged macromolecules. For example, we reported that a green fluorescent protein with a ϩ36 net theoretical charge (ϩ36 GFP) was highly aggregation-resistant, could retain fluorescence even after being boiled and cooled, and reversibly complexed DNA and RNA through electrostatic interactions.A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (18)(19)(20)(21)(22)(23)(24). We hypothesized that superpositively charged proteins such as ϩ36 GFP might also associate with negatively charged components of the cell membrane in a manner that results in cell penetration.…”

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confidence: 99%

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Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

McNaughton

Cronican²,

Thompson

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

239

260

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Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of ؉36 (؉36 GFP), can penetrate a variety of mammalian cell lines. Internalization of ؉36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When ؉36 GFP is mixed with siRNA, protein-siRNA complexes Ϸ1.7 m in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by ؉36 GFP suppresses target gene expression. We show that ؉36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of ؉36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.cell-penetrating protein ͉ nucleic acid delivery C ommercially available cationic lipid reagents are typically used to transfect nucleic acids in mammalian cell culture. The effectiveness of these reagents, however, varies greatly by cell type. A number of cell lines, including some neuron, T cell, fibroblast, and epithelial cell lines, have demonstrated resistance to common cationic lipid transfection reagents (1-4). Alternative transfection approaches, including electroporation (5) and virus-mediated siRNA delivery (6, 7), have been used; however, these methods can be cytotoxic or perturb cellular function in unpredictable ways.Recent efforts to address the challenge of nucleic acid delivery have resulted in a variety of nucleic acid delivery platforms. These methods include lipidoids (8), cationic polymers (9), inorganic nanoparticles (10), carbon nanotubes (11), cell-penetrating peptides (12, 13), cationic protein-antibody fusions (14, 15), and chemically modified nucleic acids (16). Each of these methods offers benefits for particular applications; in most cases, however, questions regarding cytotoxicity, ease of preparation, stability, or generality across different cell lines remain. Easily prepared reagents capable of effectively delivering nucleic acids to a variety of cell lines without significant cytotoxicity therefore are of considerable interest.We recently described resurfacing proteins without abolishing their stru...

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mentioning

confidence: 99%

“…A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (18)(19)(20)(21)(22)(23)(24). We hypothesized that superpositively charged proteins such as ϩ36 GFP might also associate with negatively charged components of the cell membrane in a manner that results in cell penetration.…”

mentioning

confidence: 99%

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

McNaughton

Cronican²,

Thompson

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

239

260

View full text Add to dashboard Cite

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“…Supercharged proteins are a class of engineered proteins that can be fused with the target proteins for superior membrane penetration properties [35], providing an alternative to CPP 'tags' [11]. Liu and colleagues [36] have recently demonstrated a potent delivery vehicle for proteins using supercharged GFP (+36 GFP).…”

Section: Supercharged Proteinsmentioning

confidence: 99%

Intracellular Delivery of Proteins By Nanocarriers

Ray

Lee

Scaletti

et al. 2017

Nanomedicine (Lond.)

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Intracellular delivery of proteins is potentially a game-changing approach for therapeutics. However, for most applications, the protein needs to access the cytosol to be effective. A wide variety of strategies have been developed for protein delivery, however access of delivered protein to the cytosol without acute cytotoxicity remains a critical issue. In this review we discuss recent trends in protein delivery using nanocarriers, focusing on the ability of these strategies to deliver protein into the cytosol.

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“…The basic mechanisms and applications of cationic CPPs have been widely studied [8][9][10][11][12]. Recent studies have reported the preparation of superpositively charged variants of green fluorescent protein (GFP) by mutating acidic amino acid residues (Glu and Asp) on the solvent-exposed surface to cationic amino acids residues (Arg and Lys), without a loss of proper folding [13][14][15][16]. Although wild-type GFP cannot permeate cells, superpositively charged GFPs can pass through cell membranes because of their highly positive two-dimensional cationic cluster on the solvent-exposed protein surface.…”

Section: Introductionmentioning

confidence: 99%