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66 Cell-Penetrating Peptides: Processes and Applications
The general localization pattern of cell-delivered streptavidin obtained by fluorescence
and electron microscopy is very similar. However, the results of electron
microscopy studies suggest that, in parallel with endocytosis, another poorly understood
process governs transportan-aided cellular translocation of proteins. Electron
microscopy studies reveal also that conglomerates containing several labeled streptavidin
and biotinyl–transportan molecules can cross the plasma membrane and enter
the cells, showing the applicability of peptide-mediated transport for big supramolecular
complexes.
3.7 METHODOLOGICAL CONSIDERATIONS
3.7.1 CARGO–CPP CONJUGATION STRATEGIES
Several types of conjugation strategies have previously been used including pHsensitive
linkers, the reduction-sensitive disulfide bond, and direct backbone fusion.
As CPPs have a cellular distribution and possible biochemical actions of their own,
a labile bond between them and a cargo molecule would be advantageous. However,
for larger cargo sizes in which the uptake rates are lower, the extracellular stability
of the disulfide bond could be too low and a stable bond could be more favorable.
Consequently, we have chosen to use disulfide bonds for smaller molecules and
stable cross-linkers for larger cargo.
3.7.2 DISULFIDE HETERODIMERS
For synthesis of unsymmetrical disulfides between transport peptides and Cyscontaining
cargo, the 3-nitro-2-pyridinesulphenyl (Npys) derivative of Cys was coupled
to the ε-amino group of Lys 13 in transportan, or N terminally in the other CPPs.
The S-Npys-protected peptides have been shown to react rapidly with thiols to form
disulfides. 35 Because the ring nitrogen acts as an internal base in the reaction, it can
be performed in low pH, which suppresses homodimer formation.
The constructs were synthesized using different solvent systems. However, a
deoxygenated mixture of 3:2:1 DMSO/NMP/0.1 M sodium acetate, pH 4.5, gave
the highest yield for peptides and PNA. The mixture was stirred gently overnight
under nitrogen gas. In the case of peptide or PNA–CPP constructs, purification was
performed on the reverse phase HPLC column and the final yield of the construct
was around 30 to 50%.
3.7.3 CONJUGATION OF TRANSPORTAN TO PROTEINS
For synthesis of the GFP–transportan construct, covalent protein dimers were
reduced by incubating purified recombinant GFP with DTT. Residual DTT and salts
were removed by gel filtration on Sephadex G-15 column. For the transportan–GFP
conjugation, several solvent systems were tested and the best was found to be
deoxygenated 40 mM Hepes buffer, pH 7.2, which contained 0.1 M KCl and 30%
(v/v) ethanol.