Smart Protein Refolding System Based on UCST-Type Ureido Polymers.

We have reported that ureido polymers exhibit upper critical solution temperature (UCST)-type phase behavior in solution, which is the opposite of lower critical solution temperature (LCST)-type behavior. Furthermore, UCST-type ureido polymers undergo liquid-liquid phase separation (LLPS) upon cooling rather than the liquid-solid phase transition of the typical LCST-type polymers. In this study, ureido polymers with hydrophobic groups were prepared to evaluate the effects of cooling-induced LLPS of UCST-type polymers on refolding of proteins. When protein was heated with a ureido polymer functionalized with undecyl groups, aggregation of the protein was prevented. Subsequent cooling incubation resulted in the spontaneous release of the protein from the polymer. The released protein had enzymatic activity, suggesting that the protein refolded properly. Interestingly, efficient refolding was observed when the solution of the UCST-type ureido polymer and protein was incubated at around the phase separation temperature of the polymer, implying that cooling-induced LLPS of the polymer enhanced the release of the protein. Additionally, by centrifugation at 4 °C, the refolded protein was readily separated from the ureido polymers, which precipitated upon cooling.

Rational Design of UCST-type Ureido Copolymers Based on a Hydrophobic Parameter.

Thermoresponsive polymers exhibiting upper critical solution temperature (UCST)-type behavior under physiologically relevant conditions have potential as biomaterials. The phase separation temperature ( Tp) of the UCST-type polymers can be increased by copolymerization with hydrophobic comonomers. Quantitative index parameters that could be used to rationally tune the Tp are lacking, however. We have reported that ureido copolymers such as poly(allylamine- co-allylurea) (AM-PU) and poly(l-ornithine- co-citrulline) exhibit UCST-type solution behavior under physiologically relevant conditions. In this study, AM-PU was hydrophobized by acylation. Tp of AM-PU can be regulated in a wide temperature range from about 20 to 80 °C or even higher by 20 mol % acylation with acetyl, propionyl, isobutanoyl, or pivaloyl groups, implying considerable impacts of hydrophobic groups on UCST phase separation. We observed a liner relationship between Tp and the hydrophobic parameter, log P, of the acyl groups. Furthermore, the acylation significantly reduced the influence of serum components on Tp by eliminating interactions of copolymers with serum components such as proteins and lipids. Acylation also abolished pH dependence of Tp which had been observed for unmodified AM-PU. Owing to the simple relationship between log P and Tp and the inertness of the acylated copolymer to serum components and pH changes, it is possible to rationally design copolymers exhibiting UCST-type behavior at a desired temperature under biological conditions.

Design of a Tunable Self-Oscillating Polymer with Ureido and Ru(bpy)3 Moieties.

An upper critical solution temperature (UCST)-type self-oscillating polymer was designed that exhibited rhythmic soluble-insoluble changes induced by the Belousov-Zhabotinsky (BZ) reaction. The target polymers were prepared by conjugating Ru(bpy)3 , a catalyst for the BZ reaction, to ureido-containing poly(allylamine-co-allylurea) (PAU) copolymers. The Ru(bpy)3 -conjugated PAUs exhibited a UCST-type phase-transition behavior, and the solubility of the polymer changed in response to the alternation in the valency of Ru(bpy)3 . The ureido content influences the temperature range of self-oscillation, and the oscillation occurred at higher temperatures than conventional LCST-type self-oscillating polymers. Furthermore, the self-oscillating behavior of the Ru-PAU could be regulated by addition of urea, which is a unique tuning strategy. We envision that novel self-oscillating polymers with widely tunable soluble-insoluble behaviors can be rationally designed based these UCST-type polymers.