Public defence in Biotechnology, MSc Jianhui Feng

Title of the thesis: Exploring Condensate Properties of Engineered Proteins in Cellular and in vitro Systems for Materials Design

Thesis defender: Jianhui Feng
Opponent: Prof. Evan Spruijt, Radboud University, the Netherlands
Custos: Prof. Markus Linder, Aalto University School of Chemical Engineering

The push for novel synthetic materials has created a particular interest in biomimetic designs utilizing proteins, a primary building block for many natural materials. Evidence suggests that liquid-liquid phase separation (LLPS) of proteins is an essential assembly step for materials such as spider silk and mussel adhesives, enabling the pre-organization of proteins into liquid condensates before they acquire more solid structures. LLPS mechanism thus offers a promising route for engineering biosynthetic materials. However, the molecular mechanisms behind engineering LLPS-enabled protein condensates and tuning their properties for materials design are not yet fully understood. 

This thesis explores whether cells could function as living test tubes for identifying and characterizing engineered protein condensates. A key focus is to investigate the extent to which the insights gained from condensate behaviors in living cells correlate with those observed under in vitro conditions. The thesis's results show that condensate formation and its properties are very sensitive to protein concentrations and environmental conditions (e.g., pH and temperature). The correlation of condensate behaviors between cellular and in vitro systems becomes stronger when the conditions are adjusted to match more closely those of the cell. In addition, we show that the spider silk protein NT2RepCT undergoes laser-induced condensate gelation and low pH-induced transition from liquid condensate to solid-like fibrillar structure in vitro, with both behaviors  recapitulated in cellular systems. Finally, by initially studying protein behaviors in living cells, we successfully identify a series of redesigned adhesive proteins that can function as LLPS-enabled underwater adhesives with strong bonding strength. 

Overall, we show that intracellular characterization of condensate properties could be used as an initial means to understand and modify condensate properties in vitro for engineering materials properties. We also highlight some critical aspects that need to be considered when using this method for materials development.

Keywords: Liquid-liquid phase separation, biomolecular condensates, spider silk, underwater adhesives, intracellular characterization, protein engineering

Thesis available for public display 7 days prior to the defence at Aaltodoc. 

Contact information: 
jianhui.feng@aalto.fi