PhD Studentship: Investigating Signal Peptide Functionality for Bioprocessing

University of Birmingham

Biopharmaceuticals are a class of drug products comprising relatively complex molecules such as recombinant proteins. Many biopharmaceuticals are made in the bacterium E. coli because of its long history of safe use, well-characterised physiology and relative simplicity [1]. The range of post-translational modifications that E. coli can perform is limited, but disulphide bonding of proteins is possible. In order to do this, proteins must be translocated to the periplasm of E. coli where the disulphide bond formation machinery resides.

There are three major mechanisms by which proteins are translocated to the periplasm in E. coli: the post-translational SecA pathway; the co-translational SRP pathway; and the Tat pathway. The former two pathways translocate the polypeptide chain in an unfolded state through a pore one amino acid at a time. The SecA and SRP pathways are industrially used for periplasmic targeting of recombinant proteins.

Polypeptide chains are directed to the SecA or SRP pathways by an N-terminal signal peptide which interacts with the inner bacterial membrane and translocation machinery. Selection of the optimal signal peptide for translocation of a recombinant protein is not a straightforward task. Not only do the amino acids of the signal peptide have to correctly interact with the bacterial inner membrane and translocation apparatus, but codon usage and the mRNA secondary structure conferred by the signal peptide can also have a large role in conferring signal peptide function [3,4].

In addition, the rate at which the polypeptide chain is translocated through the inner membrane must be matched to the rate of translation, otherwise cytoplasmic accumulation will occur, potentially leading to protein misfolding. Finally, there is no such thing as a universal “optimal” signal peptide; a preferred signal peptide must be selected for each recombinant protein.

We will investigate the relationship between the signal peptide sequence (nucleotide and peptide) and characteristics in terms of protein translocation and productivity. Signal peptide performance will be tested at multiple levels using different techniques. Effects on bacterial physiology, viability and physical characteristics (eg surface charge, zeta potential, hydrophobicity) will also be monitored to ensure that signal peptides do not negatively affect the ability of bacteria to grow to a high cell density and be processed industrially. The overall outcome of the project will be a better understanding of the multiple effects of signal peptide structure on bacterial physiology, protein production and protein translocation.

 Excellent students are invited to apply, with an Undergraduate Honours degree with a minimum classification of a 1st or equivalent. Applicants should have a biosciences, chemical engineering, bioengineering, biotechnology or chemistry background and be interested in interdisciplinary research. Informal enquiries should be sent to Dr Tim Overton: t.w.overton@bham.ac.uk  

References 

1. Overton TW. (2014) Drug Discov Today. 2014 19:590-601.

2. Tsirigotaki A, et al. (2017) Nat Rev Microbiol. 15:21-36.

3. Power PM, et al. (2004) Biochem Biophys Res Commun. 322: 1038-44.

4. Zalucki YM, et al. (2009) Trends Microbiol. 17: 146-50.

Funding Notes

This project is offered as part of the BBSRC-funded Midlands Integrative Biosciences Training Partnership (MIBTP), along with the Universities of Warwick and Leicester. This scheme involves a first year comprising training courses, two miniprojects and a professional internship. The PhD project runs from years 2-4. Funding is available for UK and EU students. More details are available at http://www2.warwick.ac.uk/fac/cross_fac/mibtp/

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Type / Role:

PhD

Location(s):

Midlands of England