Cyclical, Cell-Penetrating, Peptide-Protein Fusions
Kerwin, John Forbes
Bentley, William E
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The expression of recombinant proteins often exploits amino acid motifs that can provide unique properties for recognition, post-translational modification, binding capacity, and translocation potential. Implementing a poly-histidine tag, for example, creates a unique binding site for efficient purification of a target protein from unwanted impurities. This tag can later be cleaved rendering the functional ‘native’ protein. Inteins are natural, “protein ligators”, facilitating the formation of a covalent bond between two flanking protein fragments (exteins) of a translated protein sequence. The intein excises itself upon ligation of the flanking exteins. Split-inteins can flank a target protein and upon an excision event, they enable a post-translational mechanism that cyclizes the protein by cleaving out the flanking intein sequences. Cell-penetrating peptides (CPPs) permit the translocation of exogenous macromolecules across the cell membrane, while maintaining membrane integrity. Nucleic acids, proteins, and small molecules can be linked to the CPPs through a covalent bond or a non-covalent interaction to facilitate their translocation into the cell. The uptake of a CPP-protein fusion is typically rapid and follows first-order transduction kinetics. CPP-mediated uptake can be rapidly measured by quenching extracellular fluorescence with trypan blue. Trypan blue will penetrate cells with compromised membranes that could transduce proteins unmediated. In this study, eGFP is expressed alongside the transactivator of transcription (TAT) peptide derived from HIV-1 that can function as a cell-penetrating peptide. The TATeGFP fusion protein is also expressed alongside an artificial split-intein system. The cyclized TATeGFP protein exhibits enhanced in vitro stability during protein expression, resistance against exopeptidase digestion, and resistance against chaotropic agent degradation. A cell-penetrating peptide-protein fusion can transduce a cell membrane to deliver an intracellular protein with low cytotoxic effects. A cyclical, cell-penetrating, peptide-protein fusion is a novel tool for fluorometric reporting studies and a backbone for the delivery of intracellular therapeutic proteins with enhanced thermal, enzymatic, and chemical stability.