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44 Cell-Penetrating Peptides: Processes and Applications
2. Treat the peptide with an equimolar amount of TCEP and incubate for
5 min at room temperature (TCEP should not be in excess).
3. Mix the solution with an equimolar amount of vector.
4. Incubate for 1 h at 37°C.
5. Keep 2 µg for gel analysis and store the coupled product at –80°C in
aliquots. If desired, the coupled product can be purified by HPLC.
6. Dilute 2 µg of the coupled product in 1 × SDS sample buffer (final
concentration). Run the sample on 20% SDS-PAGE without reducing
agent. The same amount of the coupled product diluted in 1 × reducing
SDS sample buffer or vector and peptide alone can be run as control.
7. Stain the gel with Coomassie blue. The yield of the reaction is routinely
above 50%.
2.5.2.1.3 Direct Linkage
The cargo can be chemically synthesized in tandem with the vector; 60 this method
is possible when the cargo is a peptide or a chemical molecule (fluorochromes,
biotin). However, once internalized, an s–s coupled peptide is likely to be separated
from the vector because of the reducing intracellular medium. This is not the case
for a chimerical molecule.
2.5.2.1.4 Fusion Proteins in Bacteria
Fusion peptides comprising the AntpHD or penetratin peptides can be expressed in
E. coli and purified. AntpHD fusion protein can be purified on heparin columns.
2.5.2.1.5 Decoupling Reaction
Incubation of cells with the decoupled product is a useful control for both fluorescent
and nonfluorescent oligonucleotides. Alternatively, oligonucleotide or vector alone
can be used.
Solution
1. 1 M cysteine (MW 121.2): dissolve 1.2 g cysteine in 10 ml distilled water.
Steps
1. Incubate the coupled product for 15 min at 37°C with 1 mM DTT (final
concentration). If the cargo or vector alone is used as control it can be
incubated with a tenfold molar ratio of cysteine in order to prevent terminal
SH group reaction with free SH present at the cell surface.
2.5.2.2 Internalization and Detection of Vector–Cargo Fusion Molecules
Procedures are basically the same as those described for vector alone except that
the cargo can be labeled for detection in place of the vector. As oligonucleotides
and peptides, PNAs fused to AntpHD and penetratin can be visualized after internalization
in cells when coupled to a fluorochrome 66 or to a biotin residue. One
advantage of the vector is that internalization efficacy is almost insensitive to all
common culture media (RPMI, DMEM-FI2) and unmodified by the presence of