Mutagenesis modulates the uptake efficiency, cell-selectivity, and functional enzyme delivery of a protein transduction domain

Abstract

Alanine scanning mutagenesis of a recently reported prostate cancer cell-selective Protein Transduction Domain (PTD) was used to assess the specific contribution each residue plays in cell uptake efficiency and cell-selectivity. These studies resulted in the identification of two key residues. Extensive mutagenesis at these key residues generated multiple mutants with significantly improved uptake efficiency and cell-selectivity profiles for targeted cells. The best mutant exhibits ~19-fold better uptake efficiency and ~4-fold improved cell-selectivity for a human prostate cancer cell line. In addition, while the native PTD sequence was capable of delivering functional fluorescent protein to the interior of a prostate cancer cells, only modest functional enzyme delivery was achieved. In contrast, the most potent mutant was able to deliver large quantities of a functional enzyme to the interior of human prostate cancer cells. Taken together, the research described herein has significantly improved the efficiency, cell-selectivity, and functional utility of a prostate cancer PTD.

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

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Resurfaced cell‐penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

“…To determine uptake efficiency we fused each of our polycationic resurfaced nanobodies to GFP and measured uptake by flow cytometry. 3T3 cells were first treated with 10–500 nM polycationic resurfaced nanobody‐GFP fusion, then washed with a phosphate buffered saline solution containing 20 U/mL heparin sulfate—which has been previously shown to remove cell surface bound protein especially supercharged proteins . Following treatment with trypsin, which has also been shown to remove and/or degrade surface bound protein, intracellular levels of nanobody‐GFP was measured by flow cytometry.…”

“…Thus, most current protein drugs and basic research tools target disease‐relevant receptors that reside on the surface of the cell or the extracellular matrix. Efforts to unlock the full potential of proteins in biomedical applications by enabling potent and functional cell penetration have been a major focus of modern biologics research . Incorporation of polycationic linkages—such as polyarginine—has previously been described as a means to enable cell penetration of various cargo, including proteins .…”

Engineered M13 Bacteriophage Nanocarriers for Intracellular Delivery of Exogenous Proteins to Human Prostate Cancer Cells

“…More recently, we described mutagenesis efforts resulting in a T7A mutant of Ypep (termed Ypep2, herein) which exhibits dramatically improved prostate cancer cell-selectivity and cell-penetration efficiencies. 41 When Ypep2 is fused to the N-terminus of p3 on phage, dramatically improved potency of phage uptake is observed in PC-3 cells, compared to phage that display Ypep. Interestingly, the potency and selectivity of phage cellpenetration is dictated by multivalency effects.…”