Protein Crystallization from Metastable Protein-Rich Droplets as a Novel Protein Purification Method
Dougay, Joel
Dougay, Joel
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2025-05-19
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Abstract
Protein purification is a critical step in protein downstream processing. Although chromatography is the most employed technique for protein purification, novel strategies that reduce operational costs and increase the amount of purified protein must be developed. These strategies can reduce the price of protein-based pharmaceutical and biotechnological products. Preparative protein crystallization is one such economically sustainable alternative to chromatography. However, protein crystallization is slow and difficult to implement in protein purification protocols. In recency, an increasing number of reports have shown that aqueous protein solutions exhibit liquid-liquid phase separation (LLPS). LLPS is a metastable phase transition that is typically induced by cooling protein aqueous samples below a well-defined temperature. In our lab, we explore how LLPS can be used to promote protein crystallization for applications as a protein purification method.
Previous work has shown that cooling aqueous samples of lysozyme protein at pH 7.4 in the presence of salt (NaCl, 0.15 mol L?1) and an organic buffering molecule, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.10 mol L?1) below the LLPS temperature produces extensive lysozyme crystallization. However, the actual yield of crystallization was not characterized. This thesis will examine the effect of temperature on LLPS-mediated crystallization, the specific additives used in our solutions LLPS, crystallization yields, and temperature-turbidity phase diagrams. This thesis will also demonstrate that LLPS-mediated crystallization is an effective alternative to preparative chromatography and protein crystallization.