Abstract
Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (–30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (–30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (–30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.
Highlights
Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics
While cationic lipids are routinely used to transfect polyanionic nucleic acids into mammalian cells, they have not been widely used for protein delivery, as proteins vary widely in their ability to be encapsulated by cationic lipids[11]
While (–30)GFP enables cationic lipids to deliver a variety of fused cargo proteins[12], its discovery from protein engineering efforts unrelated to delivery[13] suggests that more potent anionic proteins that mediate lipid-based protein delivery may exist
Summary
Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (–30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. Correspondence and requests for materials should be addressed to Proteins including genome editing agents represent an increasing proportion of biomedical research tools and new human therapeutics[1] Due to their inability to spontaneously cross the lipid bilayer, current uses of protein tools and therapeutic agents mostly target extracellular components[2]. To impart nucleic-acid-like properties onto proteins, we previously demonstrated that fusing an anionic protein, such as an engineered (–30)GFP with many surfaceexposed negative charges, to a protein of interest enables its efficient encapsulation and delivery into mammalian cells[12]. The non-mammalian origin of (–30)GFP will likely result in immunogenicity, potentially compromising the safety or efficacy of using this protein in vivo[14]
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