Fluorescence Correlation Spectroscopy Reveals Efficient
Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins
Version 3 2018-10-24, 04:17
Version 2 2018-09-27, 18:52
Version 1 2018-09-27, 18:49
Posted on 2018-10-24 - 04:17
New methods for delivering proteins
into the cytosol of mammalian
cells are being reported at a rapid pace. Differentiating between
these methods in a quantitative manner is difficult, however, as most
assays for evaluating cytosolic protein delivery are qualitative and
indirect and thus often misleading. Here we make use of fluorescence
correlation spectroscopy (FCS) to determine with precision and accuracy
the relative efficiencies with which seven different previously reported
“cell-penetrating peptides” (CPPs) transport a model
protein cargothe self-labeling enzyme SNAP-tagbeyond
endosomal membranes and into the cytosol. Using FCS, we discovered
that the miniature protein ZF5.3 is an exceptional vehicle for delivering
SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve
a cytosolic concentration as high as 250 nM, generally at least 2-fold
and as much as 6-fold higher than any other CPP evaluated. Additionally,
we show that ZF5.3 can be fused to a second enzyme cargothe
engineered peroxidase APEX2and reliably delivers the active
enzyme to the cell interior. As FCS allows one to realistically assess
the relative merits of protein transduction domains, we anticipate
that it will greatly accelerate the identification, evaluation, and
optimization of strategies to deliver large, intact proteins to intracellular
locales.
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Wissner, Rebecca
F.; Steinauer, Angela; Knox, Susan L.; Thompson, Alexander D.; Schepartz, Alanna (2018). Fluorescence Correlation Spectroscopy Reveals Efficient
Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins. ACS Publications. Collection. https://doi.org/10.1021/acscentsci.8b00446
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AUTHORS (5)
RW
Rebecca
F. Wissner
AS
Angela Steinauer
SK
Susan L. Knox
AT
Alexander D. Thompson
AS
Alanna Schepartz
KEYWORDS
fluorescence correlation spectroscopy250 nMprotein ZF 5.3Protein Cargoprotein transduction domainsCPPFCSFluorescence Correlation SpectroscopyAPEXenzymeZF 5.3SNAP-tagcytosolic protein deliveryCell-Permeant Miniature Proteins New methodscytosolic concentrationintracellular localesEfficient Cytosolic Delivery