Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pI target proteins

Abstract

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Figure 1

Phage-based ELISAs of M13-KO7 phage wrapped with polymers (A) 1, (B) 2, (C) 3 and (D) 4 binding to either DNase or BSA (negative control). Wrapping the phage (1 nM) with a 1000-fold molar excess (1 µM) of polymers 1, 2 or 3 was sufficient to reduce or eliminate non-specific binding to target DNase. Polymer 4, however, required a 10⁵ molar excess (~100 µM) to wrap the phage effectively. Throughout this report, error bars indicate standard error (n = 2–3).

Figure 2

Phage-based ELISAs of M13-KO7 phage wrapped by polymers (A) 1-trunc and (B) 1-ext. A fixed concentration of M13-KO7 phage (0.5 nM) was incubated with the indicated concentrations of each polymer, and exposed to either DNase or the blocking agent BSA for assay. Polymer 1-ext (23 amine functionalities) at high concentrations (~100 µM) resulted in high levels of cross-linked phage to DNase, but polymer 1-trunc exhibited only moderate cross-linking at the same polymer concentrations.

Figure 3

Phage wrapping ELISAs of M13-KO7 phage mixed with polymer 1 (1 µM). (A) Wrapped M13-KO7 phage abrogated non-specific binding to target DNase at phage concentrations ≤2 nM. However, binding between phage and DNase was observed in the absence of polymer 1 and at high phage concentrations. (B) Non-specific binding of M13-KO7 phage to lysozyme was also abolished by wrapping with polymer 1.

Figure 4

Phage-based ELISA comparing the phage wrapping efficiencies of peptide Lys₂₀ with polymers 1-ext and 2. The indicated phage concentrations were incubated with 10 µM of Lys₂₀ and polymer 1-ext, and 3 µM of polymer 2, before testing for binding to DNase. Polymer 1-ext resulted in the most efficient wrapping, as this wrapper eliminated non-specific binding to the target at typically problematic phage concentrations (≥5 nM).

Figure 5

Binding affinity for DNase by phage-displayed (A) DNase-1 and (B) DNase-2, and specificity for other proteins by phage-displayed (C) DNase-1 and (D) DNase-2 wrapped with 1-ext. In these phage-based ELISAs, the unwrapped phage bound strongly and nonspecifically to DNase. However, wrapping the phage with 1-ext (2.5 µM) dramatically lowered non-specific binding, and, particularly for DNase-2, strong binding to DNase was retained. In a phage-based ELISA examining specificity of binding by phage-displayed ligands, both DNase-1 and DNase-2 preferentially bound DNase. In this experiment, n.d. indicates “not determined.” However, other experiments performed under identical conditions (not shown) revealed no interaction between M13-KO7 phage (both wrapped and unwrapped) and BSA.

Figure 6

ELISA demonstrating high affinity binding of synthetic peptide DNase-1 to DNase. The DNase-1 peptide was N-terminally biotinylated for detection with HRP-conjugated streptavidin. Addition of 1 µM polymer 1 to the peptide solution resulted in a marginal effect in DNase binding.

Chart 1

Phage-wrapping polymers in pH 7 buffer.