Recombinant Expression and Purification of Ferritin-Based Core Protein Scaffolds to Promote Liquid-Liquid Phase Separation

Abstract

Understanding material assembly in nature can assist in engineering new materials that have exceptional properties similar to those of spider silk or insect resilin. These biological materials are assembled by liquid-liquid phase separation (LLPS): triggered by changes in the physicochemical environment, a solution of soluble protein condenses into a separate liquid-like protein rich phase (droplets). Over time and directed by the environment, droplets solidify into their final functional structure. To study LLPS inside cells, the Corelet system (core scaffolds to promote droplets) was previously developed and published. In this system, cells are genetically modified to produce recombinant proteins that form protein droplets reversibly in the presence of blue light. The recombinant proteins consist of two fusion proteins: the ferritin heavy chain FTH1 module and the intrinsically disordered protein region IDR module. The FTH1 mod-ule contains human ferritin heavy chain and spontaneously assembles to form nanoparticles in solution. In the presence of blue light, the IDR module associates with the ferritin core (nano-particle) triggering phase separation. In other words, the FTH1 module forms a “scaffold” that the IDR module can associate with when a blue light turns on. This triggers droplet formation. The aim of the thesis is to take the first step in applying the Corelet system to study drop-let formation outside cells. To do this, a recombinant overexpression (Escherichia coli) and purification method is developed to obtain enough protein to study material formation in isolation. This cannot be done with the original methods used to observe droplet formation inside cells as it is a low-expression system. The thesis work was done in three parts: (1) screening for suitable conditions for both soluble and insoluble expression, (2) refolding the protein purified from the insoluble fraction, and (3) biophysical characterization to assess function. The thesis work focuses primarily on producing the FTH1 module. Only the preliminary assessment for the IDR module was done. The results indicate that while the FTH1 module can be obtained from the soluble fraction of the cell lysate, it is mainly found in the insoluble fraction. The total protein yield purified from the soluble fraction of the cell lysate was 5.4 mg/g of cells, however this is an overestimate because it could only be partially purified (includes degradation products and impurities). The total protein yield from the insoluble fraction was 11.1 mg/g of cells and it was successfully purified. The assembled and most likely functional ferritin core, however, was achieved only from the soluble fraction as the refolding of the protein isolated from the insoluble fraction was aggregated. However, the insoluble fraction remains an intriguing option to gain functional protein due to the high protein content. More work must be done to develop the refolding conditions. In the case of the IDR module, preliminary experiments suggest it is degraded during expression. In the future, the expression system for the IDR module must be further developed. Once this is done, then the function of the overall system (light-switch) can be tested. Overall, this work represents the first step in establishing a system to study the formation of biological materials outside the cell triggered by a reversible system controlled by light. This will impact both our understanding of materials and provide new tools to develop advanced materials from proteins.