Engineering spatial segregation within bacterial hosts for bio-therapeutic protein production
Spatial segregation of metabolism in bacteria is achieved by localising enzymes inside of sophisticated proteinaceous organelles called bacterial microcompartments (BMCs). In the past decade significant progress has been made to engineer these BMCs, in particular the 1,2-propanediol utilising (Pdu) BMC, to permit the construction of synthetic microcompartments within the bacterial cell that have potential to be used as novel nano-bioreactors.
Research objectives The overall aim of this project is to develop an alternative method to traditional disulphide bond formation approaches (folding in the eukaryotic endoplasmic reticulum and in the periplasm of prokaryotes) by engineering compartmentalisation within the bacterial cytoplasm of bacteria such as E. coli which will allow for an environment that promotes correct protein folding and disulphide bond formation of recombinant proteins.
To achieve this, proteins of interest will be compartmentalised by fusing them to targeting peptides which direct the proteins to a microcompartment shell. Both the shell and the targeting peptides are derived from the Pdu BMC from Citrobacter freundii. The student will look to develop the components of the microcompartments through interplay with components from other BMC systems. For instance, one of the subclasses of the glycyl radical enzyme-containing microcompartments (GRMs) is predicted to form a shell composed of fewer types of proteins compared to the Pdu BMC. The candidate will have the opportunity to investigate if these other BMC shell proteins can interact with the Pdu system we are using to advance our available tools.
Applying the compartmentalisation technology, we will initially determine if a protein of interest forms its disulphides in the absence of an active catalyst when targeted to a microcompartment shell, shielding it from the cytoplasmic reducing pathways. Then catalytic enzymes will be introduced to help the formation of native disulphide bonds. Subsequently, this will be extended for the production and purification of bio-therapeutic proteins such as granulocyte colony stimulating factor (GCSF), bovine pancreatic ribonuclease, N-terminus of tissue inhibitors of metalloproteinase (N-TIMP), and antibody fragments such as Fab to Hepatitis B and TNF inhibitors. We will also investigate the feasibility at which these folding systems can be produced in industrial hosts and how they can aid product purification at low costs.
Research Training The candidate will develop skills across several interdisciplinary areas including microbiology, molecular biology, enzymology, protein biochemistry ensuring broad training in molecular cloning, multi-protein assemblies, synthetic biology, imaging (TEM) and protein structure determination (X-ray crystallography)
Person specifications This PhD project is suitable for a candidate with a Molecular Biology, Biochemistry, Biotechnology, Biochemical Engineering, or Chemical Engineering Bachelor or Masters degree.
Funding Available to UK and EU applicants resident in UK for over 3 years
Start of a project: March 2017
Apply now through PRISM online https://www.prism.ucl.ac.uk/#!/project/209/view. Application deadline is 15th January 2017
Funding available to UK and EU applicants resident in UK for over 3 years