Engineering of heterobifunctional biopolymers for tunable binding and precipitation of Strep‐Tag proteins and virus‐like nanoparticles

Abstract Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin‐like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of any Strep ‐tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding‐induced multivalent crosslinking upon protein capture. The efficient biotin‐mediated dissociation of the bound Strep‐ tag II fusion protein from the streptavidin‐ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus‐like particle (VLP)‐like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding‐induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP‐like E2 protein nanoparticle as a surrogate.