Affinity purification of multifunctional polymer nanoparticles

Abstract

We report that multifunctional polymer nanoparticles approximately the size of a large protein can be "purified", on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The selection process takes advantage of the thermoresponsiveness of the nanoparticles allowing "catch and release" of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (K(dapp) ≈ nM) and a narrower affinity distribution than the average of particles before purification (K(dapp) > μM) at room temperature but can release the peptide just by changing the temperature. We anticipate this affinity selection will be general and become an integral step for the preparation of "plastic antibodies" with near-homogeneous and tailored affinity for target biomacromolecules.

Figure 1

a, Amino acid sequence of melittin. Hydrophobic and positive charged residues are printed in green and red respectively. b, The chemical structures of functional monomers used for NP synthesis. c, Solution phase AFM images of NPs. A height profile of cross-section (light blue line) is shown in insert.

Figure 2

a, Protocol for affinity sorting. Randomly copolymerized NPs are incubated with melittin immobilized agarose beads in 100 mM phosphate buffer (pH7.4) at 25 °C. Ratio of NPs remaining in solution after incubation with different volumes of melittin immobilized beads is plotted in (b). Interaction (QCM) between melittin and NPs before incubation (yellow) and remaining after incubation (gray) is shown in (b) insert. NPs on the beads were first washed with water at 25 °C then eluted with cold water (1 °C). Each cycle (12 hour incubation) was repeated until no further elution of NPs was observed. Fluorescent intensity of NPs in each fraction from washing (red) and cold elution (blue) cycles is plotted in (c) and (d) respectively. Interaction between melittin and wash fraction in 100 mM phosphate buffer (red circles) and in water (red triangles) at 25 °C are shown in (c) insert. Interaction between melittin and NPs before incubation (yellow) and cold elution (blue) in water at 25 °C are shown in (d) insert.