Intracellular uptake of peptides and anti-Ras scFv fusion proteins and their anti-proliferative activity.

<p>(A) Schematic representation of peptide-anti-Ras scFv cDNA constructs. DNA encoding peptides and the Y13-259-scFv cDNA were fused as described in Materials and Methods and cloned into the <i>Nco</i>I and <i>EcoR</i>I sites of pET21c. The white boxes represent V_H and V_L of the Y13-259 scFv sequence. The round box indicates the sequence encoding the peptides: Tat or BR2. The <i>Nco</i>I and <i>EcoR</i>I restriction sites and stop codon positions are also indicated. (B) Protein uptake was analyzed by Western blotting of fractionated lysates from HCT116 cells treated with PBS, anti-Ras scFv, BR2- or Tat-scFv fusion protein (each, 2 µM) at 37°C for 2 h. An anti-His antibody was used to detect intracellular Tat-scFv and BR2-scFv (28-kDa). (C) The anti-proliferative activity of peptides and anti-Ras scFv fusion proteins. HCT116 cells were exposed to the indicated concentrations of anti-Ras scFv, Tat- or BR2-scFv fusion protein at 37°C for 24 h. Cell proliferation was determined using the MTT assay. Data represent the mean ± s.d. of three independent experiments.