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Transportans 63
Commonly, the protein of interest is expressed in bacteria as a fusion with the
cell-penetrating sequence and the construct is purified by applying different chromatographic
methods. However, if the pure protein is available in sufficient amounts
or its expression as a fusion protein with CPP sequence is cumbersome (e.g.,
antibodies), then chemical cross-linking of protein to a delivery peptide is a good
alternative. Moreover, more than one CPP molecule could be coupled per molecule
of the protein of interest by the chemical cross-link, which could facilitate the cell
entry of bigger proteins. However, there are some drawbacks as well. The delivered
protein cannot be dissociated from the transporter in the cells and the modification
of functional domains of proteins by the cross-linker could lead to decrease or even
loss of activity or function.
We chose TRITC-labeled avidin as the first medium-sized protein. The construct
was prepared from avidin–TRITC and Cys-( N,ε Lys 13 )transportan by first coupling the
cross-linker to the amino groups of avidin and then linking transportan via the sulfhydryl
groups. The cellular uptake of the resulting TP–avidin–TRITC construct was rapid; in
5 min the red fluorescent signal was detectable in the plasma membrane of COS-7 or
Bowes melanoma cells. After 30 min, staining was detectable all over the cytosol. It
was mainly localized granularly, but some diffuse staining was present as well.
Modification of antibodies with transportan following the same cross-linking
protocol enabled them to translocate into cultured cells. The time scale of internalization
and the cellular distribution of transportan-conjugated antibodies were analogous
to those of avidin–transportan constructs. We derivatized polyclonal antibodies
recognizing human mitochondrial NifS protein with transportan. The ability of the
antibodies to recognize the antigen was not lost upon conjugation with the delivery
peptide, since detection of NifS protein by transportan-modified antibodies was not
compromised in Western analysis. However, whether the cell-transduced antibodies
are competent to find and bind their intracellular target and thereby to modulate its
activity is not clear so far. We may conclude that transportan can carry relatively
big proteins with molecular mass at least up to 150 kDa across the plasma membrane
into the cell interior.
The possibility of utilizing a noncovalent complex of the transport peptide and
a cargo molecule for delivery has not been studied so far. To fill this gap, we took
advantage of the capability of avidin and streptavidin to form an extremely stable
complex with biotin or biotin-labeled molecules. Streptavidin alone or in combination
with the unlabeled cell-penetrating peptide cannot traverse the plasma membrane
(Figure 3.3B). However, coapplication of biotinylated transportan along with
streptavidin Texas Red conjugate into the culture medium led to intense staining of
the plasma membrane of the cells in 5 to 15 min, confirming that, in solution,
biotinylated transportan associated with labeled streptavidin (Figure 3.2b) and targeted
the formed complexes into the plasma membrane. At 15 min, but more
markedly at 30 min and later, transportan–streptavidin complexes had relocated from
plasma membrane into cortical cytoplasm and were detectable in the perinuclear
area of a cell (Figure 3.3A).
Internalized streptavidin–Texas Red (or its complexes with biotinyl–transportan)
gave rise to weak diffuse staining of the cytosol with strong punctuate accumulation,