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64 Cell-Penetrating Peptides: Processes and Applications
FIGURE 3.3 (Color Figure 3.3 follows p. 14.) (A) Delivery of streptavidin-Texas Red
conjugate (SA-TxR) by biotinyl-transportan (bTP) into COS-7 cells. The cells were incubated
for 2 h at 37°C with 2 µM biotinyl-transportan and streptavidin-Texas Red (red) (1:100
dilution) in IMDM. (B) Nonbiotinylated transportan was applied into the culture medium
along with SA-TxR. The plasma membrane was visualized with concanavalin A-FITC (green)
(2 µg/ml). (C) Delivery of anti-biotin monoclonal antibody into Bowes cells by biotinyltransportan.
Cells were incubated with 2 µM bTP and 1 µg/ml anti-biotin antibody (clone 33
“Boehringer–Mannheim”) in culture medium for 2 h at 37ºC and stained after fixation with
anti-mouse-FITC (“Sigma” 1:100). (D) Uptake of bTP-SA-TxR at 0°C (2 h incubation).
indicating that part of the delivered proteins was probably confined to vesicular
substructures or concentrated in specific regions or organelles. The cellular distribution
of streptavidin–Texas Red that had been translocated into the cells as a
noncovalent complex with biotinyl–transportan was, in general, very similar to that
observed for covalent transportan–avidin–TRITC constructs. We may conclude that
covalent coupling of the transport peptide to a cargo protein is actually not obligatory,
and their strong complexing is also sufficient for efficient transduction of a protein
into cells.
Streptavidin has exceptionally high affinity towards biotin and forms very stable
complexes with biotinyl–transportan. However, how strongly the delivery peptide
must associate with a protein in order to guarantee transfer of a protein from outside
to inside the cells is not known. Antibiotin monoclonal antibodies bind biotin with
affinity several orders of magnitude lower than streptavidin does. Therefore, we
investigated whether the interaction between the biotin moiety of transportan and