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Penetratins 31
2.4 DELIVERY OF HYDROPHILIC CARGOES INTO LIVE CELLS
WITH PENETRATIN VECTORS
Since first reports demonstrating the efficient cell delivery of hydrophilic molecules
linked to penetratin or AntpHD, 31-33 several applications have been developed thanks
to the Antennapedia homeodomain, its third helix, and several variants (for reviews see
References 28, 29, 34, and 35). Penetratin and penetratin-derived peptides have been
used successfully to deliver chemical molecules, proteins, oligopeptides, oligonucleotides,
and peptide-nucleic acids into live cells, in vitro and in vivo. The next paragraphs
describe the mode of linkage and highlight some typical applications. A more comprehensive
list of cargoes delivered by this vector system is presented in Table 2.3.
2.4.1 PRINCIPLES OF CARGO–VECTOR LINKAGE
One of the first applications has been to link and internalize oligonucleotides bearing
a free thiol group to the cysteine naturally present in the β-turn between the second
and the third helix of AntpHD. 36 AntpHD was also used to deliver recombinant
polypeptides of different lengths (Table 2.3). In that case, the cargo sequences were
fused to the C terminus of AntpHD and the fusion proteins expressed in Escherichia
coli were purified thanks to the heparin-binding properties of the homeodomain.
However, most of the experiments were achieved with the penetratin-1 peptide. The
three main types of coupling protocols used are illustrated in Figure 2.2.
In a first protocol, penetratin is synthesized with an additional N-terminalactivated
cysteine, protected by a nitro-pyridinium group that prevents peptide
homodimerization and facilitates disulfide bond formation with the reactive thiol present
on the cargo. This method is advantageous in that the cargo is released free in the
cytoplasm as a result of disulfide bond breakage in the reductive cytoplasmic milieu.
In a second method, the cargo and vector are chemically synthesized in continuity.
In that case, no coupling reaction is necessary. These first two methods also
allow the internalization of modified peptides like phospho-peptides or peptide
nucleic acid (PNA). Another possible modification is biotinylation; this modification
is useful to follow the peptide in the cells and to purify any molecule interacting
with internalized cargoes.
The third method consists in the preparation of fusion polypeptides by in vitro
recombination. The fusion is expressed in E. coli and purified easily due to the
heparin-binding properties of the third helix. Alternatively, the fusion protein can
incorporate a poly-histidine or GST sequence for purification on ion columns or
gluthathion, respectively. Indeed a tag (HA, myc, etc.) and a protease site allowing
removal of the GST moiety can be added to the construction. Because of low cost,
this is a method of choice when long cargoes are needed.
2.4.2 VECTORIZATION WITH ANTPHD
2.4.2.1 AntpHD-Mediated Internalization of Oligonucleotides
The first applications were developed with the entire homeodomain. AntpHD has
been used to internalize antisense oligonucleotides against the β-amyloid precursor