A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles

Abstract

In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al., 2017). To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency). Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

Nanotechnologies for Intracellular Protein Delivery: Recent Progress in Inorganic and Organic Nanocarriers

“…Interestingly, live cell imaging demonstrated that NPSCs delivered their protein cargo through a direct 'membrane-fusion' mechanism, avoiding endosomal entrapment [38]. The same group recently reported interesting results with 2 nm Au NP functionalised with arginine-presenting ligands (ArgNPs) to electrostatically interact with proteins presenting an oligo-glutamic acid negative tag (E-tag) in their C-term ends [40] (Figure 3C). Hence, via these carboxylate-guanidinium electrostatic interactions, complex hierarchical assemblies of ArgNPs and five types of E-tagged proteins with different molecular weights, isoelectric points (prothymosin-α (PTMA), GFP, granzyme A, Cre recombinase, histone 2A (H2A) were formed generating 350 nm range protein-ArgNPs structures.…”

Ionic polypeptide tags for protein phase separation

“…Analogous sequences and predictive rules for polypeptides that promote complex coacervation have yet to be established. The few reported examples of anionic polypeptide tags have shown the ability of ionic tags to drive the assembly of protein materials and their potential use for encapsulating enzymes and intracellular protein delivery 16,17. The work herein develops design criteria for ionic polypeptide tags that promote protein complex coacervation and characterizes the phase behavior of model protein-tag fusions.…”

Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery

“…To limit the impact of mutagenesis on protein activity while maximizing changes that assist protein delivery, short sequences have been appended to the N- or C-terminus of native proteins. These polypeptide tags have been genetically added and can confer a range of properties including supercharging, − the ability to complex with divalent metal ions, − and specific cellular localization. ,− Charged tags have been added as an alternative to surface supercharging. Following the initial demonstration that supercationic GFP’s facilitate cytosolic delivery, GFP was supercharged via C-terminal fusion with an elastin-like polypeptide .…”

“…Anionic supercharging facilitates complexation through electrostatic interactions between the protein of interest and a cationically charged nanoparticle. This approach has been used to encourage uptake into lipid-based nanocarriers, ,− ,,,, polymeric nanoparticles, ,,,,,, and inorganic nanoparticles. ,,,,,, Lipid-based nanoparticles, including phospholipid and cationic lipids are a common choice for intracellular delivery of various cargo. In a recent representative example, a branching poly glutamate tag, termed E-tag, was covalently attached to KRas and saporin to encourage encapsulation in 60 or 120 nm lipofectamine 3000 nanoparticles.…”

“…This approach has been used to encourage uptake into lipid-based nanocarr i e r s , 1 8 , 9 5 − 9 7 , 1 2 6 , 1 2 9 , 1 3 7 , 1 6 8 p o l y m e r i c n a n o p a rticles, 14,94,118,120,124,133,169 and inorganic nanoparticles. 12,13,71,72,132,170,171 Lipid-based nanoparticles, including phospholipid and cationic lipids are a common choice for intracellular delivery of various cargo. In a recent representative example, a branching poly glutamate tag, termed E-tag, was covalently attached to KRas and saporin to encourage encapsulation in 60 or 120 nm lipofectamine 3000 nanoparticles.…”