title: Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.
abstract: Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the 'cytosol' of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells.
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Kenneth K Y
authors.initials: KK
authors.lastname: Ho
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Jin Woo
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Institut des Biomolécules Max Mousseron, UMR 5247, CNRS-Université Montpellier-ENSCM et Université d'Avignon et des Pays de Vaucluse, Avignon, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Durand
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Sagardip
authors.initials: S
authors.lastname: Majumder
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.|Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.|Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America.|Biophysics Program, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Allen P
authors.identifier: http://orcid.org/0000-0002-0309-7018
authors.initials: AP
authors.lastname: Liu
mesh_terms: D058045:Artificial Cells / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D002474:Cell-Free System / Q000737:chemistry*; D005898:Glass / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D044085:Microfluidics / Q000379:methods; D010316:Particle Size; D011142:Polyvinyl Alcohol / Q000737:chemistry*; D066329:Protein Aggregates; D011663:Pulmonary Surfactants / Q000737:chemistry; D012997:Solvents / Q000737:chemistry*
chemical_list: D002457:Cell Extracts; D066329:Protein Aggregates; D011663:Pulmonary Surfactants; D012997:Solvents; D011142:Polyvinyl Alcohol