Phase transition of recombinant fusion protein assemblies in macromolecularly crowded conditions

Abstract

Artificial cells, synthetic analogues of living cells in both structure and function, have emerged as valuable tools for investigating the principles of life and for advanced applications. These artificial cells are constructed through the self-assembly of biological molecules and have been examined under macromolecularly crowded conditions. The dense environment of the intracellular cytoplasm or extracellular matrix is recapitulated by using macromolecules such as polyethylene glycol (PEG), with concentrations that typically range from 10 to 40% w/v. We investigate the self-assembly of recombinant fusion proteins composed of functionally folded globular protein and elastin-like polypeptide (ELP) into coacervates or vesicles to develop a potential artificial cell platform. Herein, we focus on understanding the phase transition of the recombinant fusion protein assemblies and vesicles in PEG-rich, macromolecularly crowded conditions by experiments and molecular dynamics simulations. It has been found that self-assembled protein vesicles undergo agglomeration in macromolecularly crowded conditions, where individual vesicles cluster to form larger aggregates. This is followed by the vesicle-to-coacervate transition and phase separation in the protein-rich particles. The PEG concentrations that induce the phase transition of the protein assemblies depend on the relative number of globular proteins at vesicle surfaces. This knowledge would be useful to provide engineering strategies for artificial cells in cell-like macromolecularly crowded conditions.