In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers.

Heather E Findlay,Nicola J Harris,Paula J Booth

doi:10.1038/srep39349

Abstract

Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks.

Highlights

Biological membranes are complex environments, with both integral and peripheral membrane proteins surrounded by bilayers composed of many different types of lipid
In most Major Facilitator Superfamily (MFS) transporters active transport is driven by an electrochemical gradient, a proton gradient in the case of LacY
In this study we present the first example of a secondary transporter incorporated into a Droplet Interface Bilayer system resulting in active transport of a molecular substrate against a concentration gradient

Summary

Introduction

Biological membranes are complex environments, with both integral and peripheral membrane proteins surrounded by bilayers composed of many different types of lipid. A variety of lipid structures have been used to replicate the membrane environment; from bicelles[1,2] and nanodiscs[3,4] to larger liposomes and giant unilamellar vesicles[5,6] These vesicles provide a self-contained inner compartment that allows for the study of movement of molecules across the bilayer and are increasingly used as drug delivery systems in the medical field[7]. There have been very few uses of integral membrane proteins in DIBs. Existing examples have predominantly been channels, including the mechanosensitive channel MscL that provides a large non-selective pore[15]. In this study we use LacY as a model protein for the incorporation of a secondary transporter into a Droplet Interface Bilayer system, using both exogenously expressed, reconstituted protein and in vitro transcription/translation to synthesise protein in the droplet

Methods

Results

Conclusion