Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Abstract

a b s t r a c tOne of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.

Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

“…After exposure of the cells to GFP, fluorescence signals were not detected inside the cell body. This finding is in line with the results of Pesce et al [ 31 ], who did not observe cell-associated fluorescence after incubation of A549 cells with GFP for 24 h. The lack of cell-associated GFP signals might be due to the fact that GFP is not internalised by the cells. Alternatively, GFP fluorescence might be quenched by the low pH value present in endocytic vesicles or lysosomes or degraded by proteolytic enzymes.…”

A Hypothesis-Free Sensor Array Discriminates Whiskies for Brand, Age, and Taste

“…We investigated GFPs fused to unfolded supercharged polypeptide chains. 27,28 These genetically engineered tags consist mainly of the pentapeptide repeat [GVGXP] n , where X is either a positively charged lysine (K) residue or a negatively charged glutamic acid (E). 29 These motifs were multimerized to exhibit 36 charged amino acids.…”

Supercharged Proteins and Polypeptides

“…Similarly generic is the fusion of a folded cationic supercharged protein to another protein to enhance cell uptake. The same holds true for fusing supercharged unstructured polypeptides to a target protein to achieve high yields of internalization: Pesce et al reported a new strategy to enhance the cellular uptake of exogenous proteins with SUP tails . The best uptake was observed with GFP‐K72 fusion, containing 72 Lys residues per tail.…”

“…All these features are far beyond what is possible with conventional polyelectrolyte polymers. Supercharging proteins and polypeptides thus provides materials with exciting properties such as hyper‐temperature resistance and the ability of overcoming biological barriers in vivo . Moreover, these highly charged structures allow the assembly of bioliquids, organelle‐like condensed matter architectures, artificial biological nanocontainers, and interfacial coatings among many others.…”