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Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers

Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers
Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers
Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) PEG 8000 and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can therefore be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channels
1932-1058
14108
Friddin, Mark S.
d3e84f8a-c24b-49b9-908c-c3fe612b39b0
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
de Planque, Maurits R.R.
a1d33d13-f516-44fb-8d2c-c51d18bc21ba
Friddin, Mark S.
d3e84f8a-c24b-49b9-908c-c3fe612b39b0
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
de Planque, Maurits R.R.
a1d33d13-f516-44fb-8d2c-c51d18bc21ba

Friddin, Mark S., Morgan, Hywel and de Planque, Maurits R.R. (2013) Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers. Biomicrofluidics, 7 (1), 14108. (doi:10.1063/1.4791651).

Record type: Article

Abstract

Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) PEG 8000 and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can therefore be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channels

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Published date: January 2013
Organisations: Nanoelectronics and Nanotechnology

Identifiers

Local EPrints ID: 347761
URI: https://eprints.soton.ac.uk/id/eprint/347761
ISSN: 1932-1058
PURE UUID: 0afb04bd-a19f-45dd-bd9f-74b1d47cc02f
ORCID for Hywel Morgan: ORCID iD orcid.org/0000-0003-4850-5676
ORCID for Maurits R.R. de Planque: ORCID iD orcid.org/0000-0002-8787-0513

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Date deposited: 30 Jan 2013 11:20
Last modified: 20 Jul 2019 00:59

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Author: Mark S. Friddin
Author: Hywel Morgan ORCID iD

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