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.
Highlights
Artificial cells are cell-like entities that mimic certain properties or functions of natural cells. [1, 2] Building artificial cells, a natural extension of biochemical reconstitution, provides a complimentary approach to reductionist cell biology to uncover cellular design principles.[3]
We found that the aggregates were caused by high protein concentration in the mammalian HeLa cell-free expression (CFE) and high poly(vinyl) alcohol (PVA) surfactant concentration required for the formation of double emulsion templated vesicles
As a yet untapped area of encapsulating mammalian CFE system as the ‘cytosol’ of lipid membraneenclosed artificial cells, we report a systematic study to examine the effect of PVA surfactant on mammalian CFE-containing double emulsion templated vesicles
Summary
Artificial cells are cell-like entities that mimic certain properties or functions of natural cells. [1, 2] Building artificial cells, a natural extension of biochemical reconstitution, provides a complimentary approach to reductionist cell biology to uncover cellular design principles.[3]. We found that the aggregates were caused by high protein concentration in the mammalian HeLa CFE and high PVA surfactant concentration required for the formation of double emulsion templated vesicles.
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