Expression of recombinant membrane proteins in Escherichia coli for therapeutic use

Secondary Supervisor(s): Dr Alan Goddard (Biosciences, Aston); Dr Alexander Darlington (Engineering, Warwick)

University of Registration: Aston University

BBSRC Research Themes:

• Renewable Resources and Clean Growth (Industrial Biotechnology)
• Understanding the Rules of Life (Microbiology, Systems Biology)

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 cellular chaperones and the components of the Sec translocon have been manipulated, producing strains which have increased capacity for membrane protein production. To complement 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 Professor Alan Goddard at Aston University (80% of project time) and the computer modelling with Dr Alexander Darlington (School of Engineering, The University of 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.

[3] 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.

[4] 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.

Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli

Secondary Supervisor(s): Dr Ewan Ross

University of Registration: Aston University

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Systems Biology)

Project Outline

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrhoeal disease in industrialized and non-industrialized countries and has been shown to be the cause of travellers’ diarrhoea and persistent diarrhoea in children and HIV patients. EAEC strains cause disease by binding to the human gastric mucosa and establishing a thick mucoid biofilm, damaging tissue by secreting various protein toxins [1]. In spite of the widespread occurrence of EAEC strains and their impact on human health little is known about how EAEC strains control the expression of genes required to establish infection and cause disease. Furthermore, EAEC strains are becoming increasingly resistant to many clinical antibiotics, decreasing the options available to treat infections [1-4]. Thus, there is a great need to understand EAEC pathology and devise new methods to combat this important group of bacterial pathogens.

In many EAEC strains, it has been shown that the expression of important virulence determinants (e.g., the attachment adherence fimbriae (AAF) required for colonization, the anti-aggregative protein dispersin (Aap) and its dedicated secretion system) is controlled by the AggR transcription factor, the master regulator of virulence [1-3]. Recently, we have been working with EAEC strains isolated from Egyptian and Brazilian children with diarrhoea [3,4]. In these strains, we have found a new version of AggR that appears to control gene expression differently. Thus, the main aim of this project will be to determine how this new version of AggR controls virulence gene expression in these strains and determine the genes that it regulates. We have already had the genomes of many of these strains fully sequenced, and so part of this project will be to analyse their genomes, to determine each strains characteristics (e.g., antibiotic resistance gene profile, virulence determinants and plasmid replicons etc.). Once key virulence and antibiotic resistance genes have been identified, the promoters that control their expression, will be amplified by PCR and cloned into reporter gene expression plasmids. The transcription regulation of these promoters will then be examined under different environmental conditions to determine how and when the expression of these important proteins is switched on. Coupled with this, the project will also examine the binding of AggR throughout each strain’s genome, using techniques such as chromatin immunoprecipitation (ChIP-seq) and RNA-sequencing (RNA-seq) to understand where AggR binds and the genes it controls. The binding of AggR will be then confirmed using in vitro biochemical techniques, such as band shift assays (electrophoretic mobility shift assays (EMSA)). The final aim of this project will be to use synthetic biology to engineer different versions of AggR that can block specific promoters and disrupt virulence gene expression in EAEC strains. Using either bacteriophage or plasmid conjugation, these new AggR alleles will be delivered to EAEC pathogens as potential therapeutic agents to combat EAEC infection.

Methodologies

This project will provide complete training in many essential bioinformatic and molecular biology techniques, which will include, PCR, DNA cloning, site directed mutagenesis, DNA sequencing, bacterial strain manipulation, reporter gene enzyme assays, ChIP-seq, RNA-seq and various in vitro biochemical techniques (band shift assays (EMSA)/ DNAse I footprinting assays).

References

1) Harrington, S.M., Dudley, E.G. and Nataro, J.P. (2006) Pathogenesis of enteroaggregative Escherichia coli infection. FEMS Microbiol Lett. 254(1):12-8. Review.

2) Yasir, M., Icke, C., Abdelwahab, R., Haycocks, J.R., Godfrey, R.E., Sazinas, P., Pallen, M.J., Henderson, I.R., Busby S.J.W. and Browning, D.F. (2019) Organization and architecture of AggR-dependent promoters from Enteroaggregative Escherichia coli. Molecular Microbiology. 111:534-551.

3) Abdelwahab, R., Yasir, M., Godfrey, R.E., Christie, G.S., Element, S.J., Saville, F., Hassan, E.A., Ahmed, E.H., Abu-Faddan, N.H., Daef, E.A., Busby, S.J.W. and Browning, D.F. (2021) Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea: similarities and differences. Virulence 12:57-74

4) França, F.L.S., Wells, T.J, Browning, D.F., Nogueira, R.T., Sarges, F.S., Pereira, A. C., Cunningham, A.F., Lucheze, K., Rosa, A.C.P., Henderson, I.R. and das Graças de Luna, M. (2013) Genotypic and phenotypic characterisation of enteroaggregative Escherichia coli from children in Rio de Janeiro, Brazil. PLOS ONE 10.1371/journal.pone.0069971.