Smart Protein Refolding System Based on UCST-Type Ureido Polymers

Abstract

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.

Spontaneous Liquid Droplet-to-Gel Transition of Citrulline Polypeptide Complexed with Nucleic Acids

“…We have reported that ureido polymers such as poly­(allylurea), − poly­(vinylurea), and poly­(2-ureidoethyl methacrylate) form liquid droplets under physiological salt and pH conditions. The droplets dissolve above the phase separation temperature ( T p ), and cooling below T p results in liquid droplets.…”

Encapsulation in cooling-induced coacervates augments DNA enzyme activity

“…10 Practical applications using the complex coacervate require strategies such as chemical crosslinking to stabilize the coacervate. 11 We previously reported that ureido polymers such as polyallylurea, [12][13][14] polyvinylurea, 15,16 and poly(2-ureidoethylmethacrylate) 17,18 exhibit LLPS over wide ranges of pH and salt concentrations including in physiologically relevant conditions. As the ureido polymers form simple coacervates, we reasoned that these coacervates would be useful in biological media.…”

“…Moreover, LLPS properties of the ureido polymers are thermosensitive. [12][13][14][15][16][17][18] The coacervate droplets formed by ureido polymers dissolve upon heating, and the coacervates form again when cooled below the phase separation temperature (T p ); T p is dependent on molecular weight and ureido content. Due to this unique upper critical solution temperature (UCST)-type thermoresponsive, ureido polymers can capture proteins in coacervates upon cooling 13 and can induce a switch between monolayer and spheroid cell culture upon a change in temperature.…”