Spatio-Temporal Photoactivation of Cytotoxic Proteins

Abstract

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Keywords: barnase; inteins; light-activation; photocaging; saporin.

Figure 1

An intein is inserted to perturb the normal protein fold and activity; a photocage (orange) is attached to a cysteine to prevent premature intein activity while a cell‐penetrating peptide (CPP) allows entry into cells.

Figure 2

Construction, splicing and cell uptake of intein‐interrupted mCherry‐CPP proteins. (A) Cell penetrating peptide sequences. (B) Flow cytometry of mCherry construct uptake in HeLa cells (values are the man of triplicate measurements of three independent experiments, error bars indicate SD). (C) Viability of HeLa cells treated with mCherry constructs (10 μM) for 2 hours. (D) Confocal microscopy showing the middle section from a z‐stack of images taken from cells incubated with mCherry constructs.

Figure 3

Characterization of delivery system in HeLa cells. (A) Flow cytometry analysis of HeLa cells after light activation of photocaged intein‐split mCherry‐HA2‐cTAT. Box plot shows the mean fluorescence with error bars representing the minimum and maximum value. All individual values are shown in the plot; ***denotes P<0.0001 in unpaired t‐test. (B) Celltiter Blue viability assay of HeLa cells treated with photocaged intein‐split mCherry‐HA2‐cTAT, intein‐split saporin‐HA2‐cTAT, intein‐split barnase‐HA2‐cTAT and control cells. Box plot shows the mean cell viability with error bars representing the minimum and maximum value. All individual values are shown in the plot. ns denotes no significance, * denotes P<0.1, and *** indicates P<0.001 in unpaired t‐test.