CASE Project: Cell-Free production of membrane protein targets underpinning agrichemical development

University of Registration: 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.

Sensing the impossible, an engineering biology approach

Project description

The Challenge

Engineering biology and its place in sensing

Imagine if we could tap into the natural magic of sensing! Creatures big and small respond to various signals, be it light, taste, odour, or even drugs. Think about sniffer dogs with their powerful noses – what if we could borrow that amazing power without actually needing the dog?

A long-term challenge of Engineering Biology (EB) (also known as synthetic biology) has been to harness natural systems to develop new and improved sensing systems. For example, one might be able to use cellular receptors to monitor hormones like insulin to control a disease, or detectors would be used to monitor different aspects of the food supply chain from detecting plant or animal diseases through to monitoring food spoilage. Alternatively, real neuronal receptors could be used to detect narcotics like fentanyl providing a detector that mimicked the human brain.

Our strategy to achieve this lofty ambition involves a “bottom up” approach where individual biological components, or small groups of components, are combined to produce a sensing element. This has the advantage of exploiting the unique capabilities of biological molecules without ethical issues associated with using GM organisms in the environment.

In our laboratory we have assembled an ensemble of methods and component systems that we have shown can be used to produce sensing systems of demonstrable global importance. These include pure samples of receptor proteins (including GPCRs) which bind and respond to specific chemical signals.

We have also developed a novel nanoencapsulation system (SMALPs) which stabillises these receptors and allows the development of sensing nano-particle that combines the GPCR with other elements required to generate a readable signal. We have also established connections with agricultural, medical and defence sectors who are interested in applying these sensors to real world problems.

The Scientific Background

The project proposed focuses on our development in 2009 of a novel method that allows us to make and stabilise membrane proteins that nature uses in sensing (1). The method (SMALPs) uses a polymer to encapsulate a sensing membrane protein complete with it’s surrounding lipid environment. We have shown that this is a generically applicable method that produces very stable and active samples of a wide range of membrane proteins. In this project our aim is exploit our previous work which shows that SMALPs can be used to make G-protein Coupled Receptors (2). GPCRs are “nature’s detector molecules” and have evolved to bind to an exceptionally wide range of ligands (including hormones, cell signalling molecules and neurotransmitters. Ligand binding induces these proteins to change shape. We have shown that we can detect this shape change (3) using fluorescence providing the route to a revolutionary new detection system based on GPCRs.

Work in the Project

The project will continue the work of a current MIBTP PhD student who has developed a production system for a range GPCR-SMALPs. These include hormone receptors, messenger receptors and a receptor for the active ingredient in cannabis. This new project will use these reagents to test different detection modalities including the use of fluorescence and electrochemical detection. The project will aim to develop a platform approach to harnessing these GPCR-SMALPs for an entirely new generation of molecular detectors.

References

1. Knowles, T.J., et al., J Am Chem Soc, 2009. 131(22): p. 7484-5.
2. Grime R.L. et al.,Nanoscale. 2021 Aug 21;13(31):13519-13528,
3. Routledge S. J. et al., Biochim Biophys Acta Biomembr. 2020 Jun 1;1862(6):183235

Techniques

• Engineering Biology “Design, Build, Test” approaches
• Membrane protein expression, extraction and purification
• Biophysics (including fluorescence, absorbance, circular dichroism, analytical ultracentrifugation, dynamic light scattering).
• Imaging including electron and fluorescence microscopy
• Polymer chemistry
• Robotics automation and control
• Assay design and development