Ion channels are membrane proteins of interest for medical research and drug discovery, however a major bottleneck in obtaining functional measurements is the requirement to over-express the channel in-vivo. Cell-free (CF) protein expression is an alternative in-vitro approach capable of expressing proteins from a supplied DNA template - the method is fast, requires minimal apparatus and can be stabilised for the expression of membrane proteins by the addition of lipids or detergents. One drawback is the expense of commercial CF systems, however this can be economised by performing the reaction in microdroplets. This is attractive as microdroplets immersed in lipid-oil can be manipulated into contact to form a lipid bilayer, potentially allowing for ion channel expression and characterisation to be fully coupled. This study addresses the feasibility of achieving this goal by first investigating the stability of interdroplet bilayers formed in the presence of pre-incubated CF systems. Under these conditions the bilayers failed in <10 min, however a combination of diluting the mixture and adding vesicles was found to enable measurements of >30 min. The CF expression of the small prokaryotic potassium channel KcsA was then verified, in addition to the pore domain region of the eukaryotic hERG channel, where 20 ng/ul-74 ng/ul was expressed depending on the reaction conditions. Single-channel currents were subsequently obtained in interdroplet bilayers formed directly from the CF mixture, indicating that the channels were capable of self-inserting into the bilayer for measurements in both cases. The findings of this study support the feasibility of coupling the CF expression and electrical characterisation of ion channels in microdroplets and represent a progression toward the development of a high-throughput platform for screening novel pharmaceutical compounds.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628789 |
| Date | January 2014 |
| Creators | Friddin, Mark Samuel |
| Contributors | De Planque, Maurits |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/369412/ |
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