A Universal Tag To Supercharge Proteins for Cytosolic Delivery

Supercharged proteins are some naturally occurring proteins that bear an abnormally high number of charged amino acids but can fold correctly and function normally. Owing to the nanoscale three-dimensional charge distribution associated with folding, the superpositively charged proteins possess the ability to rapidly enter mammalian cells with a potency much greater than cell-penetrating peptides and protein transduction domains. To harness the advantage, some proteins have been extensively mutated at their surface-exposed residues to generate "supercharged" variants such as +36GFP. Despite its effectiveness, this approach is time-consuming and only applicable to a few proteins that can tolerate the massive mutagenesis. Here, we proposed a tagging strategy to supercharge proteins and expand its application for the cytosolic delivery of proteins. Using the pan-protein binding ability of Coomassie brilliant blue (CBB), we developed a CBB-bearing cationic peptide CBB-R8 that can anchor on the protein surface and supercharge proteins without mutagenesis. The charge density of tagged proteins can be finely tuned by varying the tag/protein molar ratio, thus achieving a superior delivery efficiency to the cytosol than the canonical +36GFP. Mechanism studies revealed that the tagged protein could instantly and directly access the cytosol with little participation of endocytic pathways, resulting in the majority of delivered proteins being located in the cytosol. Moreover, the tagged proteins were discharged in the cytosol and return to biological functions after delivery, capable of catalyzing, blocking, and manipulating diverse targets inside cells. In conclusion, this simple plug-and-charge methodology greatly facilitates the preparation of supercharged proteins and expands its application to more intracellular targets.