2Lek Pharmaceuticals, Ljubljana, Slovenia
Libraries of phage-displayed random peptides are routinely used to identify target-binding peptides. Phages are commonly eluted in a nonspecific manner, especially if there are no available ligands of the particular target to use as competitors. However, the present study clearly demonstrates that nonspecific elution is not always able to break peptide-target interactions. To circumvent this we have developed an improved nonspecific elution strategy that uses ultrasound to release target-bound phages and enables selection of high-affinity clones in a single step.
Phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even nonprotein targets (1,2,3). A typical round of selection involves: (i) target immobilization; (ii) reaction of the library with the target; (iii) removal of unbound phages through multiple washes; and (iv) elution of bound phages from the target followed by their amplification in bacterial cells. Several rounds of screening are performed until the increase in phage output, or binding assays performed with phage pools, indicates that the population of binding phages has been adequately enriched (4,5).
Typically, target-bound virions are released by nonspecific elution with acidic buffer such as 0.2 M glycine-HCl, pH 2.2 (1). Extreme pH and other means of nonspecific elution (e.g., high ionic strength, reductants such as DTT, specific proteases) tend to loosen the phage-target interaction, or denature or degrade either the target or the phage-displayed peptide. In cases where ligands of particular target are known and available, competitive elution may be preferred. Alternative methods of competitive, but nonspecific, elution involve using the free target molecule as an eluant (1,6) or adding bacterial host directly to the target-bound phages (7).
For a nonspecific elution strategy to result in successful affinity selection, the essential condition is thought to be appropriate washing stringency. Washing protocols can be optimized empirically for every single target. Under controlled washing conditions any target-unrelated binders should be removed, whereas selective binding of phages to the target should not be compromised (3,7).
Most commonly, the concentration of surfactants in the washing buffer is raised and more washing steps are performed in successive rounds of selection when higher stringency works in favor of affinity selection. Ionic strength or pH can also be varied and even known ligands of the target can be added to the washing buffer (7,8,9,10).
Here we show that the main reason for unsuccessful selection of ligands from phage-displayed libraries with nonspecific elution may not be the poorly chosen washing conditions but rather the inability of the nonspecific elution method to break the strongest virion-target interactions.
Sonication has been used to break the antigen-antibody interaction. Digoxin was removed from immobilized anti-digoxin antibody (11). The oscillation of cavitation bubbles produced by high-power ultrasound is known to cause hydrodynamic sheering stress, which aids in dissociation of complexes. To overcome the limitation of nonspecific elution methods in phage display we propose a completely new approach, based on the use of ultrasound.Materials and Methods Selection Protocols
Microtiter plates (StreptaWell, Roche, Mannheim, Germany) precoated and preblocked with streptavidin (Sigma-Aldrich, Steinheim, Germany) were incubated with an aliquot of 2 × 1011 phages (random cyclic heptapeptide phage display library, Ph.D.-C7C; New England Biolabs, Beverly, MA, USA), diluted to 100 µl with PBST (135 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4; pH 7.4), containing 0.1% Tween 20, for 1 h at room temperature with gentle agitation. Nonbinding phages were discarded by washing the wells with different washing strategies (Table 1). Bound clones were eluted with different elution strategies (Table 1). Washing and elution steps are described in detail below.
Eluted phages were amplified by infecting Escherichia coli ER2738 host cells (New England Biolabs). After 5 h growth at 37°C, bacteria were removed by centrifugation (12,186× g), and phages in the supernatant were precipitated by addition of 1/6 volume of PEG/NaCl solution (20% polyethylene glycol-8000, 2.5 M NaCl) and overnight incubation at 4°C. The precipitate was resuspended in a small volume of PBS and amplified eluates were titered to determine phage concentrations. Finally, eluates from the last round of selection were used to infect plated bacterial host cells and 10 plaques were selected at random. Individual phage clones were then grown and purified for analysis.Washing and Elution Steps in Protocols A–F
A brief description of washing and elution steps is given in Table 1. In Protocol A, all three selection rounds were performed in the same manner. Following 1 h hybridization of the phage library with streptavidin, unbound phage clones were removed by washing the microtiter plate surface 10 times with 0.2% PBST. Next, residual phage clones were eluted at room temperature for 1 h with 100 µl of 100 µg/mL free streptavidin solution to compete the bound phages away from the immobilized target.