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124 Cell-Penetrating Peptides: Processes and Applications
attachment did not significantly affect MTS peptide-translocating activity. However,
when the SA peptide was synthesized partly as a retro peptide, i.e., with amino acid
residues of the MTS in reverse order, the peptide failed to be imported into cells as
determined by import and functional assays. 102 The side chain topology and the
peptide amide bond direction of the MTS seem to be critical requirements for peptide
import function; either of these factors may influence the ability of the peptide to
form desirable secondary conformation for membrane interaction.
6.5 METHODS FOR ATTACHING CARGO AND TARGETING
DOMAINS TO TRANSLOCATING PEPTIDES
Translocating peptides must be linked to at least one other module to be biologically
valuable. First, they must be linked to a biologically active module, often referred
to as cargo. Second, it may be necessary to target the biologically active module to
a particular cell type or specific intracellular compartment, in which case inclusion
of a third module that allows specific recognition through a receptor–ligand interaction
is required.
In some cases it seems that the covalent linkage of cargo to translocating peptide
is not essential for efficient delivery of the cargo. For example, the plasmids pGL2
luciferase and pCMV βgal were individually mixed with a multiple antigenic peptide
(MAP) comprising eight branching units, with each unit containing the SV40 large
T-antigen NLS (TPPKKKRKVEDP) and a lysine pentapeptide acting as a cytoplasmic
translocation signal. The exposure of Chinese hamster ovary (CHO) cells to the
admixture resulted in expression of both gene products and it was proposed that the
cationic peptide associated with the negatively charged phosphate DNA backbone
via electrostatic bonds. 103 A reliance on electrostatic interaction may be adequate
for import of oligonucleotides that typically contain a negatively charged backbone;
however, uptake of proteins and peptides that, by their very nature, differ in charge
will likely always require covalent attachment to the peptide vector.
To date, one exception to this rule, in which the strength of the noncovalent
interaction between biotin and avidin was exploited, has been reported. A biotinylated
translocating peptide was synthesized and subsequently reacted with fluoresceinated
streptavidin; this vector–cargo molecule was imported into cells, whereas
fluoresceinated streptavidin alone was not. 104
There are two general methods available for linking translocating peptides to
peptide and protein cargo. First, the same method used to generate the cargo, whether
chemically by stepwise solid-phase peptide synthesis or biologically by biosynthetic
expression of recombinant protein, may be modified to include synthesis of the
translocating peptide. This is a relatively straightforward task when solid-phase
peptide synthesis is employed because the synthesis may be extended to include the
extra peptide residues at either the C or N terminus (Figure 6.2A) or coupled using
an orthogonal protection strategy to produce a peptide with a branched format. 33,34
The inclusion of a translocation peptide in the expression of a recombinant
protein requires that the plasmid encoding the protein be modified to contain the
additional nucleotides that will encode the desired peptide sequence (Figure 6.2B).