Liposome-Encapsulated <i>Escherichia coli</i> Lysates to Reconstitute Intracellular Macromolecular Crowding Effects

Intracellular macromolecular crowding impacts biomacromolecule behavior, including oligomerization, phase separation, and diffusion. However, understanding crowding effects in cells is challenging as cells respond and adapt to perturbations. Therefore, replicating in-cell crowding in liposomes would provide a good alternative to studying the consequences of macromolecular crowding. Here, we achieve physiological macromolecular crowding levels using <i>Escherichia coli</i> lysates in liposomes, as verified with a macromolecular crowding sensor. We shrink liposomes with a gradient-wise osmotic upshift to reach the high macromolecular crowding effects. We see that lysate induces higher macromolecular crowding than BSA at the same mg/mL, showing the need to use lysates to replicate in-cell behavior. We study the consequences of small cosolutes on macromolecular crowding and see that sugars and ATP modulate the lysate macromolecular crowding, implying they would also affect macromolecular crowding in cells. These artificial cells display the same crowding as <i>E. coli</i> at 220–300 mg/mL lysate and the same crowding as HEK293T at 50–100 mg/mL lysate. Hence, these artificial cells are a platform for obtaining information on physiologically relevant macromolecular crowding effects in a controlled environment.