Principal Supervisor: Dr Tim Overton, School of Chemical Engineering
Co-supervisor: To be confirmed
PhD project title: Investigating signal peptide functionality for bioprocessing
University of Registration: University of Birmingham
Biopharmaceuticals are a class of drug products comprising relatively complex molecules such as proteins. One such biopharmaceutical is insulin, responsible for control of blood sugar levels and taken as a drug by sufferers of diabetes. Several forms of recombinant human insulin for drug use are generated in the bacterium Escherichia coli. Many biopharmaceutical products are made in E. coli because of its long history of safe use, well-characterised physiology and relative simplicity . 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 (the Dsb enzymes) reside.
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 Tat pathway allows transport of folded proteins. To date, the SecA and SRP pathways are industrially used for periplasmic targeting of recombinant proteins, although the Tat system is currently under development for this purpose.
Polypeptide chains are directed to the SecA, SRP or Tat pathways by means of an N-terminal signal peptide which interacts with the inner bacterial membrane and translocation machinery. Following translocation to the periplasm, the signal peptide is cleaved by a protease. Although some information is known about the interaction of the signal peptide with the translocation apparatus, this is limited in the sphere of recombinant protein production, and 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 polypeptide chain translation, otherwise cytoplasmic accumulation will occur, potentially leading to protein misfolding. Finally, it is
clear from numerous studies that there is no such thing as a universal “optimal” signal peptide; a preferred signal peptide must be selected for each recombinant protein.
This project will investigate the relationship between the sequence (nucleotide and peptide) of signal peptides and their characteristics in terms of protein translocation and productivity. Signal peptide performance will be tested at multiple levels using different techniques, including:
· Effect on transcription activity (qPCR)
· Effect on mRNA stability & half-life (rifampicin treatment and qPCR)
· Effect on protein translocation (subcellular fractionation and SDS-PAGE / confocal microscopy of fluorescent proteins in live cells)
· Effect on protein activity (subcellular fractionation and ELISA / use of fluorescent proteins / periplasmically-active enzymes)
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 using industrial methods. 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.
- Overton TW. (2014) Recombinant protein production in bacterial hosts. Drug Discov Today. 2014 19:590-601.
- Tsirigotaki A, et al. (2017) Protein export through the bacterial Sec pathway. Nat Rev Microbiol. 15:21-36.
- Power PM, et al. (2004) Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli. Biochem Biophys Res Commun. 322: 1038-44.
- Zalucki YM, et al. (2009) Biased codon usage in signal peptides: a role in protein export. Trends Microbiol. 17: 146-50.
BBSRC Strategic Research Priority: Industrial Biotechnology and Bioenergy
Techniques that will be undertaken during the project:
- Confocal microscopy
- Flow cytometry
- Molecular biology techniques & cloning, qPCR, contact angle, zetasizer.