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

Shane Stone,Tatjana Heinrich,Paula Cunningham,Katrin Hoffmann,Scott Winslow,Kristin Priebatsch,Anna Mills,Christopher Chamberlain,Suzy Juraja,Nadia Milech,Clinton Hall,Jiulia Satiaputra,Mark Anastasas

doi:10.3390/biom8030051

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 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Highlights

Introduction βLactamase is used extensively as an independent intracellular reporter in mammalian cell assays; for example, in tracking signaling pathways [1,2,3] or viral fusions [4]
We have developed a split β-lactamase assay, where the complementation and formation of the functional β-lactamase molecule can only occur after cytosolic delivery of a cell penetrating peptides (CPPs)-cargo fusion protein
The β-lactamase internalization assay is based on the simple principle that CPP-dependent intracellular delivery of recombinant β-lactamase protein can be detected by cleavage of the CCF2 substrate in the cytosol (Figure 1a)

Summary

Results

The β-lactamase internalization assay is based on the simple principle that CPP-dependent intracellular delivery of recombinant β-lactamase protein can be detected by cleavage of the CCF2 substrate in the cytosol (Figure 1a). Error bars represent standard error of the mean conjugated SpyC_BLA in T47D cells is alsosamples dose-dependent, resulting in an increase the percentage of n independent experiments (duplicate within each experiment);. Β-Lactamase is conjugated to conventional CPPs of CPP-conjugated SpyC_BLA in T47D cells is dose-dependent, resulting in an increase in TAT_SpyT and Penetratin_SpyT (n = 1), as well as Phylomer CPPs 1746c27_SpyT, 1746_SpyT, the percentage of blue viable cells as estimated by flow cytometry. Error bars represent recombinantly expressed CPP_β-lactamase fusion protein (CPP_BLA) is dose-dependent, resulting in standard error ofinthe of n independent (duplicate samples);. CPP TAT (positive control) were expressed as direct fusions with β-lactamase The. CPP-mediated uptake of CPP_SpyT/SpyC_BLA conjugates (4 μM) was visualized in CHO-K1 cells. Bar scale is 50 μm (a–j) or 14.6 μm (k–n)

Split β-Lactamase Cytosolic Delivery Assay

Discussion

Mammalian Cell Culture

Confocal Live-Cell Microscopy

Peptides

Recombinant Protein Expression and Purifcation