Bioconjugation of Enzymes and IDPs for Development of Membraneless Organelles

Typical eukaryotic organelles use membranes formed by lipid bilayers in order to compartmentalize their functions within the cell. However, cells also contain membraneless organelles formed by intrinsically disordered proteins (IDPs) via liquid-liquid phase separation. The organelles form localized compartments that separate their contents from the environment.1 Here, this mechanism is used to generate artificial membraneless organelles that comprise a chemical reaction. An IDP, DEAD-box helicase (Ddx4), was bioconjugated to an enzyme, horseradish peroxidase (HRP), through the use of a bifunctional chemical linker, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), in order to examine if the enzyme could be incorporated in droplets and whether its activity would be affected. The conjugation of HRP-SMCC (43.4 kDa) to Ddx4 (25.6 kDa) was successful: SDS-PAGE analysis confirmed the presence of a product that was within the range of a full conjugate.