Selection and validation of peptides with HSP-binding properties.

<p>A. Peptide selection. Representative demonstration of peptide screening with recombinant HSPs using broad-range pH (3–10) isoelectric focusing (IEF) gels. Samples of 20 µg of the indicated peptides were applied at the anode and 2 µg of purified recombinant HSPs were applied at the cathode. The gradient of pH and electrophoretic directions are indicated on the left side of the gel. Abbreviations of recombinant HSP sources and horizontal lines are used to assist in sample identification in the distorted counter migrations affecting lanes 10–14. The complexes formed during counter migration are indicated at the right of the gel as “Vn-HSP complexes”. Estimates of the isoelectric focusing points of unbound gp96, HSP90 and HSP60 in the area of counter migrant distortion are indicated by red arrows (lane 10, 11 and 12). The yellow arrowhead at the bottom indicates unbound Vn peptide isoelectric focusing following counter migration against HSPs. Red arrowheads (lanes 5, 17 and 29) indicate the weakly staining salmon HSP70 (despite standardized dilution). The green arrowhead (middle of lane 11) indicates a complex with HSP90 resulting from Vn20 that has extended binding influence across preceding adjacent lanes. The green arrowhead at the base of the gel (lane 11) indicates the final focusing point of the errantly migrating Vn20. Abbreviations are as follows: Ec, <i>E.coli</i> dnaK (lanes 1, 14,26); Ad, <i>A.davidanieli</i> HSP70 (lanes 3, 15, 27); Mt, <i>M.tuberculosis</i> HSP70 (lanes 4, 16, 28); Sa, Chinook salmon HSP70 (lanes 5, 17, 29); Ra, rat HSP70 (lanes 6, 18, 30); Hu, human HSP70-1 (lanes 7, 19, 31); Bo, bovine HSP70-8 (8, 20, 32); gp78, hamster HSP70-5 (lane 9, 21, 33); gp96, canine GRP96 (lanes 10, 22, 34); h90, human HSP90 (lanes 11, 23, 35); h60, human HSP60 (lanes 12, 24, 36) and as blank lanes (lanes 13 and 25). B. Sequences of Vn96, Vn20 as well as Scrambled-Vn96 (Scr-Vn96) and their predicted 3D structures in aqueous solution using PEP-FOLD server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110443#pone.0110443-Thvenet1" target="_blank">[55]</a>. Red, blue, green, and black amino acid residues are acidic, basic, hydrophobic uncharged and other amino acid residues, respectively. Note that the Vn96 peptide favors a helical conformation. C. Validation of HSP binding by the Vn96 peptide <i>via</i> affinity pull-down of HSPs from total cell lysate. MCF-7 breast cancer cells were lysed and processed as described in experimental procedures. Streptavidin-coupled magnetic beads saturated with either biotinylated-Vn96 (b-Vn96) or biotinylated-scrambled sequence of Vn96 (b-Vn96-Scr) peptides were used to perform the pull-down assays. In the immunoblot, 1% volumes of total cell lysate were run as input proteins to compare with proteins bound by the Vn96 peptides. The heat shock proteins tested are indicated. The right lower panel shows both HSP27 (indicated as ‘>’), HSP10 (indicated as ‘<<’) and a non-specific band (indicated as ‘*’).