Leveraging lipid-protein interactions to engineer spatial organization in cell-free systems
Inventors
Peruzzi, Justin Alexander • Kamat, Neha Prashant • Steinkuehler, Jan
Assignees
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Publication Number
US-12589073-B2
Publication Date
2026-03-31
Expiration Date
Abstract
The present disclosure provides methods and compositions for controlling the localization of proteins in lipid structures. The disclosure also provides methods for selecting appropriate proteins for desired localization and distribution in lipid structures based on hydrophobic thickness.
Core Innovation
The invention relates to controlling protein localization in lipid membranes by using hydrophobic-thickness matching, described as hydrophobic mismatch, between a protein and lipid bilayer domains. A first protein having a first hydrophobic thickness is matched to a first domain having a second hydrophobic thickness so that the difference is no greater than about 5 angstroms.
A method incubates together a plasmid encoding the first protein, a cell-free protein synthesis system, and a lipid structure comprising a lipid bilayer with the first domain. The incubation is performed under conditions that promote integration of the first protein into the first domain and enable the first protein to traverse the lipid bilayer.
The disclosed approach further describes controlling membrane organization and partitioning using lipid bilayer domains and hydrophobic mismatch, including multi-protein and multi-domain arrangements and modulation associated with lipid phase separation tuned by hydrophobic mismatch. Documented examples report that hydrophobically matched membranes increase cell-free expression and proper folding, produce maximal pore-protein function when matched, and enable sorting and enrichment of vesicle populations with differential permeability.
Claims Coverage
The partial content contains one independent method claim, supported by multiple dependent claims that refine incubation temperature, lipid-structure composition, optional post-integration incubation, and additional multi-protein and multi-domain hydrophobic-thickness relationships.
Hydrophobic thickness matching between protein and lipid domain
A first protein having a first hydrophobic thickness and a lipid bilayer first domain having a second hydrophobic thickness are matched such that the difference is no greater than about 5 angstroms.
Integration and transmembrane traversal promoted during cell-free incubation
Incubating together a plasmid encoding the first protein, a cell-free protein synthesis system, and a lipid bilayer under conditions that promote integration of the first protein into the first domain of the lipid bilayer, wherein the first protein traverses the lipid bilayer.
Overall, the claims require hydrophobic-thickness matching within about 5 angstroms between the protein and a lipid bilayer domain and incubation conditions that promote integration of a cell-free expressed protein into that domain so the protein traverses the bilayer. Dependent claims further constrain temperature ranges, lipid composition, optional post-integration incubation, and multi-protein and multi-domain hydrophobic-thickness relationship rules.
Stated Advantages
Increased cell-free expression and proper folding in hydrophobically matched membranes.
Maximal pore-protein function when hydrophobic thickness is matched.
Enrichment and sorting of vesicle populations with differential permeability.
Within-single-membrane domain formation and partitioning enabled and assessed.
Documented Applications
Controlling protein localization in lipid membranes by leveraging hydrophobic mismatch between protein hydrophobic thickness and lipid bilayer domain hydrophobic thickness.
Cell-free protein synthesis in combination with hydrophobically matched membrane domains to support protein expression and proper folding.
Function testing of pore proteins using matched membranes.
Sorting and enrichment of vesicle populations with differential permeability using hydrophobic thickness matching.
Creating and assessing within-single-membrane domain formation and partitioning using lipid-protein FRET and protein-protein interaction assays.
Modulating membrane organization associated with lipid phase separation tuned by hydrophobic mismatch.
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