FIG 3.

Pgp3 binds to the antimicrobial peptide LL-37 to form stable complexes. (A) Eight micrograms of LL-37 was precipitated alone (lane 1) or with GST agarose beads (lanes 3 and 4) or GST-Pgp3 fusion protein beads (lanes 6 and 7) in a total volume of 200 μl PBS. After spinning down the beads, 10% of each bead pellet (P, lanes 3 and 6) and corresponding remaining supernatant (S, lanes 4 and 7) were loaded onto an SDS-polyacrylamide gel for electrophoresis separation, and the resolved bands were detected with an anti-LL-37 antibody by Western blotting. Note that a portion of LL-37 pulled down by GST-Pgp3 still was complexed with GST-Pgp3 after SDS denaturation. (B) A His-tagged Pgp3 mixed with various concentrations of LL-37, as indicated at the top of the figure, for 30 min at 37°C was loaded onto a native gel with 20 μl/lane for electrophoresis separation, and the resolved bands were probed with an anti-LL-37 (a) or anti-Pgp3 (b) antibody. Due to the strong positive charge of LL-37, the free LL-37 peptide (in the absence of any detergents) ran out of the native gel from the top (lane 1). The anti-LL-37 antibody detected LL-37 complexed with Pgp3, while the anti-Pgp3 antibody detected Pgp3 in trimers or high orders of oligomers, as indicated on the right. (C) Streptavidin-conjugated detection pins, after being coated with biotin-LL-37 at 5,028 nM or without coating (blank), were immersed in PBS containing tag-free Pgp3 (purified by cleaving off from the GST-Pgp3 fusion proteins) at 1,040, 2,604, or 5,208 nM or control protein GST or Pgp4 at 5,208 nM, as indicated on the right of the figure, for 300 s (association period). The pins then were moved to a fresh PBS solution for another 300 s (dissociation period). During the entire process, the molecular mass that bound to the pins was monitored using a portable BLItz system and recorded as response units as shown along the y axis.