Understanding the Coacervate-to-Vesicle Transition of Globular Fusion Proteins to Engineer Protein Vesicle Size and Membrane Heterogeneity

Abstract

Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-Z E ) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (Z R -ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.

Recent Advances in Complex Coacervation Design from Macromolecular Assemblies and Emerging Applications

“…Jang et al. [ 80 ] found that protein complexes show phase transitions from one‐phase solution, through an intermediate coacervate phase, and finally to vesicles by tuning temperature. Keating et al.…”

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

“…[232][233][234][235] For coacervates formed by amphiphilic surfactants or proteins, the transition between self-assembled coacervates and vesicles are investigated by tuning the pH, temperature and composition. [236,237] These coacervate droplets are able to sequester biological molecules, such as proteins, RNAs, magnesium ions, and DNAs, despite no bounded membrane exists. [238][239][240][241] The partition principles of the loaded macromolecules are investigated, and in turns exploited to release cargos controllably, for instance, upon pH changes.…”

“…[ 232–235 ] For coacervates formed by amphiphilic surfactants or proteins, the transition between self‐assembled coacervates and vesicles are investigated by tuning the pH, temperature and composition. [ 236,237 ]…”

Thermosensitive Vesicles from Chemically Encoded Lipid‐Grafted Elastin‐like Polypeptides

“…[19,20] Championsl ab developed another approach to access protein vesicles from ELPs,e specially using charge interactions with peptide zippers. [21,22] Francis lab also reported series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles after enzymatic cleavage of asolubilizing protein tag. [23] Natural lipidated proteins are reference models for the development of relevant artificial lipid-protein conjugates due to their diverse properties,s uch as bioactivity,s elfassembly and cell membrane anchoring properties that can regulate different cellular functions.…”

“…Kiick's lab also reported conjugates of elastin‐like and collagen‐like polypeptides (ELP‐CLP) and the production of thermoresponsive, collagen‐binding drug delivery vehicles [19, 20] . Champion's lab developed another approach to access protein vesicles from ELPs, especially using charge interactions with peptide zippers [21, 22] . Francis’ lab also reported series of fusion proteins that have been developed to self‐assemble spontaneously into stable micelles after enzymatic cleavage of a solubilizing protein tag [23] …”