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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 anyStrep‐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 boundStrep‐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. 

To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5′ cap and 3′ poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.