Principal Supervisor: Dr Tim OvertonLink opens in a new window

Co-supervisor: Doug Browning, Aston University

PhD project title: New tools for efficient recombinant protein production

University of Registration: University of Birmingham

Project outline:

Recombinant protein production (RPP) is a key tool in the production of biopharmaceuticals such as insulin as well as in basic biomedical and molecular biology research. E. coli is commonly used as a host for the production of simple proteins (Overton, 2014). However, development of RPP processes in E. coli is often problematic due to metabolic burden, protein misfolding, and stress responses that can lead to low protein yield and poor growth.

This project is a collaboration between Dr Tim Overton (University of Birmingham) and Dr Doug Browning (Aston University). We are interested in two main areas of RPP: development of new promoter systems; and periplasmic expression.

A key factor in RPP processes is control of protein synthesis, usually at the level of transcription initiation. A promoter controls expression of the gene encoding the protein of interest and use of a chemical inducer allows regulation of the promoter. Many promoters currently in use suffer from problems: some are leaky, in that they are slightly switched on even in the absence of inducer; some give poor control of expression levels; and many chemical inducers are expensive, limiting use in low and middle income countries.

Recently, we have worked on novel promoter systems to address these problems and improve control of RPP. These include:

• The PAR system, a suite of promoters designed to offer tailored expression levels permitting expression at different amounts (Hothersall et al., 2021);
• The NAR system, induced by the cheap and readily available ion nitrate (Hothersall et al., 2022); and
• A promoter system induced by urea, another cheap and readily available molecule.

We have characterised each of these promoter systems in shake flask cultures with model and commercial recombinant proteins (such as antibody fragments and growth hormones), and investigated their function at a single-cell level using flow cytometry. We have also started to investigate some of these systems in intensified bioreactor cultures; commercial RPP processes operate in large-scale bioreactors (50 000 litres and more) at high biomass concentrations (OD₆₅₀ values of 100 or more), therefore we need to understand how these systems function under these growth conditions.

A second factor we are interested in is the location of RPP. The periplasm is located between the inner and outer membrane of the Gram negative envelope. Unlike the cytoplasm, the periplasm is an oxidising environment, and is home to the disulphide bond synthesis machinery. Therefore, for recombinant proteins that require disulphide bonds for function (such as antibody fragments) the periplasm is an ideal location for targeting. In addition, the periplasm offers advantages for release and purification of proteins.

However, targeting proteins to the periplasm can be difficult. Most periplasmic export is via the Sec apparatus, whereby unfolded polypeptide chains are translocated one amino acid at a time, whereupon they fold in the periplasm. This is a slow process of limited throughput; problems can include protein misfolding in the cytoplasm or periplasm, blockage of Sec, and envelope stress. All of these can lead to lowered protein yield and poor growth.

In this project we will explore RPP in these areas. The project will be developed in conjunction with the successful student.

References

Overton, TW (2014) Recombinant protein production in bacterial hosts. Drug Discovery Today doi: 10.1016/j.drudis.2013.11.008

Hothersall J, Godfrey RE, Fanitsios C, Overton TW, Busby SJW, Browning DF. (2021) The PAR promoter expression system: modified lac promoters for controlled recombinant protein production in Escherichia coli. New Biotechnology doi:10.1016/j.nbt.2021.05.001

Hothersall J, Lai S, Zhang N, Godfrey RE, Ruanto P, Bischoff S, Robinson C, Overton TW, Busby SJW, Browning DF. (2022) Inexpensive protein overexpression driven by the NarL transcription activator protein. Biotechnology and Bioengineering doi: 10.1002/bit.28071

 

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth – Industrial Biotechnology

Techniques that will be undertaken during the project:

Techniques will be chosen based on the specifics of the project as developed by the supervisors and student. We would expect techniques from the following list:

Molecular biology / cloning

Reporter gene assays – LacZ, GFP

Flow cytometry

Protein analysis – SDS-PAGE, fractionation

Microscopy – confocal, SEM, potentially more advanced techniques (with support from BALM).

Fermentation development

Analysis of physicochemical properties of bacteria – BATH assay, zeta potential

 

Contact: Dr Tim OvertonLink opens in a new window