Screening of liquid–liquid phase separation conditions for proteins with a mixed solution kit of biomacromolecular crowding agents.

Protein liquid–liquid phase separation (LLPS) is a significant process in biology and materials research. We created a screening kit of 96 chemical formulations based on polyethylene glycol as a biomacromolecular crowding agent to determine the optimal conditions for the occurrence of protein LLPS. LLPS conditions of target proteins were discovered to provide novel ideas for intervention and therapy of LLPS-related disorders. [Display omitted] • Different solutions of the kit offer more possibilities for LLPS of protein to occur. • Screening kit can provide new conditions that promote or inhibit LLPS. • Screening kit is inexpensive, easy to use, and highly reproducible. Protein liquid–liquid phase separation (LLPS) is a significant process in biology and material sciences. Finding suitable conditions for protein LLPS is essential for its in-depth and systematic study. Here, we provide a mixed solution screening kit based on biomacromolecule crowding agents to find protein LLPS conditions. The screening kit consists of 96 chemical formulations, in which the biomacromolecule crowding agents are used as the key components. The aqueous solution of the target protein is mixed with each chemical agent in the kit and then placed in an incubator for some time. The LLPS conditions for the target protein can be found by examining the LLPS phenomena in the droplets. We used nine proteins as model target proteins—lysozyme, bovine serum albumin, bovine hemoglobin, ovalbumin, catalase, FUS, Aβ, Tau, and α-Synuclein—to evaluate the performance of the screening kit. We found that utilization of the screening kit is efficient and useful to find different conditions for LLPS of proteins. The screening results reveal two types of conditions: those that promote and those that inhibit LLPS. Both conditions are extremely valuable because those that promote LLPS can be used to do extensive studies on the LLPS processes of the proteins of interest, whereas those that inhibit LLPS can be utilized as leads for developing novel medications to treat LLPS-related disorders. Furthermore, novel applications for the kit were investigated by utilizing the screening products. [ABSTRACT FROM AUTHOR]