Induction of Liquid-Liquid Phase Separation In Vivo

Liquid-liquid phase separation (LLPS) within cells can form membrane-free organelles, which play an important role in a wide range of biological processes. LLPS are complex phenomena caused by weak multivalent interactions. LLPS can be induced by different combinations of molecules, including single proteins, multiple proteins and proteins with nucleic acids. A necessary step in the workflow of initiating the phase separation assembly and disassembly studies of biomolecular condensates is the induction of this process. Several methods have been used to induce LLPS in vitro, depending on the nature of the protein under study and the desired experimental design. A major challenge in demonstrating LLPS in vivo is having accurate metrics to unambiguously demonstrate that a specific protein or structure is indeed a phase separator in the cellular environment.

Fig. 1. Classification of proteins inducing liquid–liquid phase separation: sequential, structural and functional characterization. (Ozawa Y, et al., 2022)

Customized Services

CD BioSciences offers phase separation analysis services for purified proteins to develop high quality proteins for phase separation analysis and in vitro LLPS assays by using these recombinantly expressed and purified proteins. Depending on the properties of the protein under study, we offer several methods to induce LLPS in vitro.

• Reduce the ionic strength.
• Dilute the concentrated denaturant solution
• Add aggregates
• Change the temperature
• Remove the recombinant tag.
• Mix with conjugation partners.

Our experts have also worked to find other indicators besides overexpression to demonstrate LLPS in vivo, such as recapitulating the phase diagram in living cells. We can construct phase diagrams by systematically varying two experimental conditions to determine the conditions under which phase separation occurs. Based on phase diagram data, we can determine the role of the valence and chemical properties of proteins in the regulation of phase behavior and provide the possibility that phase separation occurs under physiological conditions. However, the ability to assess material properties is more limited in the cell. Here, CD BioSciences focuses on the induction characteristics of LLPS in vivo to explore the physical properties of biomolecular condensates. We offer the following strategies to induce LLPS in vivo.

• Increasing Scaffolding Proteins Concentration
• Inducing Dimerization and Oligomerization
• Optogenetic Reconstitution
• Knockout/Knockdown of Target Proteins

Advantages of Characterization of LLPS In Vivo

• Integrates multiple experimental approaches to provide different and often complementary information
• Reports LLPS behavior under real physiological conditions.
• Ensures the biological relevance of LLPS processes.

We offer different assays allowing us to non-invasively map the material properties of condensates within living cells. It is important to address whether these specific properties have an impact on function. We aim to develop assays that alter the properties of condensates to some extent and affect their function. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.