β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

Abstract

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 &micro;M. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Keywords: cell internalization; cell penetrating peptide; cytosolic delivery; functional validation; high-throughput screening.

Figure 1

A cell penetrating peptide (CPP)- mediated β-lactamase internalization assay. (a) Recombinant β-lactamase can be delivered into cells when conjugated to CPPs, for example through SpyTag/SpyCatcher conjugation chemistry (step i). The internalization of β-lactamase (step ii) is detected after the addition of dye substrate (step iii) through cleavage of cytosol-retained CCF2 substrate (step iv), which is measured via a change in fluorescence signal from green (520 nm) to blue (450 nm); (b) The internalization of CPP-conjugated SpyC_β-lactamase (SpyC_BLA) in CHO-K1 cells is dose-dependent, resulting in an increase in the percentage of blue viable cells as estimated by flow cytometry. Increased fluorescence is indicative of greater cytoplasmic delivery capacity of the CPP. β-Lactamase is conjugated to conventional CPPs TAT_SpyT (n = 4) and Penetratin_SpyT (n = 2), as well as phylomer CPPs 1746c27_SpyT (n = 7), 1746_SpyT (n = 5), 0084_SpyT (n = 6), and 0031_SpyT, 0167_SpyT, 0169_SpyT, 1869_SpyT, and 1788_SpyT (n = 3). Unconjugated SpyC_BLA does not internalize (‘No CPP’ negative control; n = 7). Error bars represent standard error of the mean of n independent experiments (duplicate samples within each experiment); (c) The internalization of CPP-conjugated SpyC_BLA in T47D cells is also dose-dependent, resulting in an increase in the percentage of blue viable cells as estimated by flow cytometry. β-Lactamase is conjugated to conventional CPPs TAT_SpyT and Penetratin_SpyT (n = 1), as well as Phylomer CPPs 1746c27_SpyT, 1746_SpyT, 0084_SpyT, 0031_SpyT, 0167_SpyT, 0169_SpyT, 1869_SpyT, and 1788_SpyT (n = 3). Unconjugated SpyC_BLA does not internalize (No CPP negative control; n = 3). Error bars represent standard error of the mean of n independent experiments (duplicate samples); (d) The internalization of recombinantly expressed CPP_β-lactamase fusion protein (CPP_BLA) is dose-dependent, resulting in an increase in the percentage of blue viable cells as estimated by flow cytometry. β-Lactamase is expressed fused to conventional CPP TAT (n = 3), as well as Phylomer CPPs 1746c27 and 1746 (n = 2). SpyC_BLA (negative control) does not internalize. Error bars represent standard error of the mean of n independent experiments (CPP samples in triplicate; SpyC_BLA samples in triplicate (n = 1) and duplicate (n = 2)).

Figure 2

Live-cell confocal microscopy visualizes CPP-mediated β-lactamase internalization. The CPP-mediated uptake of CPP_SpyT/SpyC_BLA conjugates (4 µM) was visualized in CHO-K1 cells (a–f) and T47D cells (g–n). SpyC_BLA was conjugated to CPP_SpyT peptides. Unconjugated SpyC_BLA (No CPP negative controls) shows minimal evidence of internalization in both CHO-K1 and T47D cells (a,g,k). Conjugation with TAT_SpyT (b,h,l) or phylomer CPPs 1746c27_SpyT (c,i,m), 1746_SpyT (d,j,n), 0084_SpyT (e), and 0169_SpyT (f) enabled CPP-dependent uptake of SpyC_BLA. The degree of uptake is CPP-dependent and visualized by blue fluorescing cells. Bar scale is 50 µm (a–j) or 14.6 µm (k–n).

Figure 3

A CPP-mediated split β-lactamase cytosolic delivery assay. (a) Live-cell functional uptake can be measured when N-terminal β-lactamase (N-BLA), fused to a CPP sequence, penetrates the cell membrane to complement C-terminal β-lactamase protein (C-BLA) in the cytosol (step i). Expression of the split β-lactamase moieties as fusions with either SpyTag (CPP_N-BLA_SpyT) or SpyCatcher (SpyC_C-BLA) facilitates rapid formation of functional β-lactamase proteins through SpyT/SpyC ligation inside the cells (step ii). This internalization is detected after addition of dye substrate (step iii) through cleavage of cytosol-retained CCF2 (step iv), which is measured via a change in fluorescence signal from green (520 nm) to blue (450 nm). (b) Internalization of CPP_N-BLA_SpyT and β-lactamase complementation in CHO-K1/SpyC_C-BLA cells is dose-dependent, measured by CCF2 cleavage (increase in % blue viable cells as estimated by flow cytometry). N-BLA_SpyT protein (No CPP negative control) shows a negligible effect on the β-lactamase complementation signal over cell-line background at all concentrations tested. Error bars represent standard error of the mean of two independent experiments (duplicate samples). (c–f) Live cell confocal microscopy visually confirms the CPP-mediated cytosolic delivery of CPP_N-BLA_SpyT (4 µM) in CHO-K1/SpyC_C-BLA cells and subsequent β-lactamase complementation signal. N-BLA_SpyT protein (c, negative control) shows minimal evidence of internalization. β-Lactamase activity is dependent on cytosolic delivery of N-BLA_SpyT with phylomer CPPs 1746c27_N-BLA_SpyT (d), 1746_N-BLA_SpyT (e), or 0084_N-BLA_SpyT (f). Bar scale is 50 µm.