Primary supervisor: Prof Tim Dafforn, University of Birmingham

Non-academic partner: Dr John Mina, Syngenta

Project description

Studies of membrane proteins have represented a historical challenge to biochemists. This stems from a lack of extraction methods that preserve the delicate structures and hence functions of membrane proteins. In the last decade there has been a revolution in the methods used to extract membrane proteins with the method based on Styrene-Maleic Acid Lipid Particles (SMALPs) being one of the leading methodologies in this movement. SMALPs can extract membrane proteins complete with the local stabilising lipid bilayer. We have shown in previous work with Syngenta that SMALPs maintain both structure and function for membrane proteins extracted from the Greenbottle fly, L. sericata. However, this project also highlighted the deficiencies of using complex tissues to generate pure protein samples, alongside challenges in developing assays which require vectoral transport of metabolites.

In this current proposal we aim to address these issues by developing a Cell Free (CF) platform for membrane protein production that resolves membrane protein production and assay challenges. The project will focus initially on agrichemical targets in L. sericata including membrane protein receptors and ion channels. We will then assess the yield and specific activity of nAChR produced using a range of commercially available CF systems alongside reagent mixes made in house. We will test these CF systems in combination with SMALPs (which provide options for structural biology) and vesicle systems (for development of transport assays).

Once a successful CF system has been established the method will be trialled with other nAChR subunits with the aim of developing a platform approach for generating receptors with differing subunit mixes for further study.

Project plan

The project is envisaged to have 3 phases:

Phase 1: Evaluation of available CF systems (commercial and generated in-house) for producing an established “test” membrane protein that includes a single transmembrane helix fused to GFP. The fluorescence signal from GFP provides a rapid method for evaluating protein production in 96 well plate format. This evaluation will also assess different polymer nano-particles (including SMA) and vesicle formats.

Phase 2: The CF systems that generate the highest yield of protein in phase 1 will then be used to produce a specific agrochemical target. The CF system will be optimised in terms of yield and specific activity (using fluorescent or radioligand binding and transport assays using vesicle systems) and a final optimal CF process established.

Phase 3: The optimised CF system will be used to produce a second homomeric and heteromeric membrane protein for study.

Candidates are encouraged to contact Prof Tim Dafforn to discuss the project before applying if they wish to.