Project Outline

Membrane proteins are essential for life, being involved in a myriad of indispensable cellular process such as energy production, solute transport and cell communication. As they are often intricately involved in the establishment of many disease states, membrane proteins can make incredibly effective drug targets [1]. However, in their host organism they are often present at very low levels and are notoriously difficult to work with, due to their hydrophobic nature. To produce enough material to work with, integral membrane proteins are often overexpressed in model organisms, such as Escherichia coli [1]. In most E. coli recombinant protein production (RPP) systems, recombinant genes are expressed from strong promoters on high copy number plasmid. However, high-level expression of membrane proteins can be toxic to cells, or substantially inhibit growth, as the expression rate and level of membrane proteins saturate the Sec translocon, which inserts membrane proteins into the E. coli membrane [2]. Low-level expression of membrane proteins can prevent this and can be achieved by manipulating various expression conditions, using specialised strains and weaker expression systems [1,3]. Previously, we have constructed a suite of new expression strains and systems that can be used to control the expression of membrane proteins to maximise their production in E. coli [3,4]. Using these new expression systems, you will trial the production of various high-value membrane proteins and generate new expression strains in which the components of the Sec translocon and translational machinery have been manipulated, producing strains which have increased capacity for membrane protein production. To completement this work, you will carry out computer-aided modelling to determine the bottlenecks that occur during membrane protein production in E. coli. The results from this will be used to inform additional strain construction and expression experiments.

The laboratory-based work for this project will be carried out with Dr Doug Browning and Dr Alan Goddard at Aston University (80% of project time) and the computer modelling with Dr Alexander Darlington (School of Engineering, Warwick) (20% project time). Complete training will be given for all molecular biology and computational work.

References

[1] Kesidis, A., Depping, P., Lodé, A., Vaitsopoulou, A., Bill, R.M., Goddard, A.D., Rothnie, A.J. (2020) Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts. Methods. 180:3-18.

[2] Collinson, I., Corey, R.A., Allen, W.J. (2015) Channel crossing: how are proteins shipped across the bacterial plasma membrane? Philosophical Transactions Royal Society B 370: 20150025.

[2] Hothersall, J., Godfrey, R.E., Fanitsios, C., Overton, T.W., Busby, S.J.W. and Browning, D.F. (2021) The PAR promoter expression system: Modified lac promoters for controlled recombinant protein production in Escherichia coli. New Biotechnology. 64, 1-8.

[3] Hothersall, J., Osgerby, A., Godfrey, R.E., Overton, T.W., Busby, S.J.W and Browning, D.F. (2022) New vectors for Urea-Inducible Recombinant Protein Production. New Biotechnology. 72:89-96.

Techniques

1. Molecular biology/synthetic biology wet work (Aston): PCR, DNA manipulation, DNA purification, DNA cloning, strain construction (P1 transduction and gene doctoring).
2. Protein expression, analysis and purification (Aston): SDS-PAGE, Western blotting, cell fractionation, membrane isolation and protein purification (affinity chromatography).
3. Computer modelling (Warwick): dynamical modelling, flux balance analysis, Python, MATLAB (Note: initial and advanced training will be provided).