(340e) Enzyme Purification and Sustained Enzyme Activity By Fusion with Phase-Separating Intrinsically Disordered Protein

In recent decades, biocatalysis using enzymes to synthesize organic molecules has become more prominent for academic studies and pharmaceutical manufacturing. Enzymes are preferred catalysts due to their high selectivity and specificity for chemical reactions, which make the overall synthesis easier to control. However, enzymes are typically used in their crude state since it is time-consuming and cost-prohibitive to purify them using conventional chromatographic methods. Impurities in the crude enzyme solutions might result in unwanted byproducts. Hence, a nonchromatographic purification method would greatly benefit biocatalytic processes. In this work, a synthetic membraneless compartment has been created with enzymes fused to an intrinsically disordered protein region—an Arg/Gly-rich domain (RGG) of LAF-1 protein from Caenorhabditis elegans P granules. The RGG domain is capable of undergoing liquid-liquid phase separation and forming liquid condensates triggered by changes in temperature or salt concentration. Leveraging this phase separation property of RGG, we purified enzymes-RGG fusions through centrifuging the liquid condensates and resolubilizing protein pellet in a new buffer. Three enzymes from different classes have been successfully fused to RGG domains—kinases, ATP-dependent ligases, and phosphorylases. We have observed that enzymes purified with our centrifugation method have an increased purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to investigate and evaluate the enzyme activities. Results from enzyme assays have indicated that enzyme fusions purified by the centrifugation method exhibit enzyme activity which is at least comparable to that of crude enzymes. To conclude, we believe that this protein purification method by centrifugation is promising in overcoming the difficulties in the development of bioseparation and biocatalysis approaches in pharmaceutical synthesis.