INTERACTION OF ARTIFICIAL-INTRINSICALLY DISORDERED PROTEIN POLYMERS WITH ENZYME-LOADED GIANT UNILAMELLAR VESICLES

Abstract

Membrane-less organelles (MLOs) are dynamic sub-cellular compartments that form primarily via the liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs). IDPs are necessary for the reconstitution of MLOs models in vitro, and may interact with curved biological membranes, or induce curvature upon binding. Cell mimicry is a sub-field of synthetic biology that aims to artificially recreate natural entities such as organelles and cells mimicking one or more functionalities of natural mammalian cells. Giant unilamellar vesicles (GUVs) constitute one model of artificial cells (ACs), and are close to mammalian cells in size and membrane composition. Notably, a new class of phase separating artificial-IDPs known as “Intrinsically disordered protein polymers” (IDPPs) were recently developed, yet were not explored for the design of GUVs loaded with artificial MLOs (AOs), nor for their plausible interactions with artificial membranes. In this work, we aim to investigate these IDPPs for such applications. To this end, we separately express and purify two versions of IDPPs (Q20 and Q30), and homogenize them in glucose buffer. Next, we stimulate homogeneous Q20 or Q30 with BSA-FITC or β-galactosidaseRho (β-galRho) or catalase-FITC (Cat-FITC) that distinctly co-condensed with IDPPs into AOs in bulk. Following the assembly of a high number of BSA-FITC-loaded GUVs via the double emulsion transfer method, and the incubation of loaded GUVs in homogeneous Q20 or Q30, we demonstrate the membrane-crossing capacity of IDPPs by the formation of membrane-associated AOs. Further advancements towards encapsulating β-galRho or Cat-FITC, and testing the reproducibility of the phenomenon with both enzymes were hindered by the insertion of β-galRho into the GUV’s membrane, and the low viscosity of Cat-FITC solution preventing its encapsulation via the double emulsion transfer method