CBMNet Proof-of-Concept and Vacation Scholarship projects awarded
Following on from our latest Proof-of-Concept and Vacation Scholarship funding calls we are pleased to announce that we have funded 6 projects.
Proof-of-Concept: Newcastle University and Ingenza – L-form technologies: a novel platform for therapeutic protein production.
Many human proteins are used for the treatment of a wide variety of diseases and many more have the potential to be developed as drugs if they can be produced in sufficient quantities. To avoid contamination with agents (prions and viruses) that can cause serious diseases, these proteins are produced in bacterial or animal cells, rather than extraction from human tissue. There is therefore a need to produce sufficient amounts of novel human proteins for preliminary analyses and, if see to have therapeutic potential, for clinical trials. Production systems based on the bacterium, Escherichia coli, is the first choice system for producing such proteins. However, about one third of all human proteins are not capable of being synthesised in E. coli production systems and alternative systems have to be used. This particularly applied to proteins that are secreted from human cells and that have disulphide bonds in their final structure. Disulphide bonds are formed after synthesis and secretion from the cell and involve the formation of bonds between two amino acid residues (cysteine) in the protein. The collaboration between Newcastle University and Ingenza is aimed at relieving known bottlenecks in the production of therapeutic proteins by using a bacterium, Bacillus subtilis, that can be switched to a wall-less state that removes a major bottleneck to protein secretion. The project involves generating such strains and evaluating their performance under commercial biomanufacturing conditions. If successful, the strains could expand the range of therapeutic proteins available for the treatment of specific diseases.
Proof-of-Concept: University of Sheffield and Excivion – Viral antigen production for diagnosis and vaccine-mediated disease prevention by rational glycoengineering-mediated protein secretion in a mammalian cell factory.
The global incidence of mosquito-borne flavivirus induced disease such as dengue has increased exponentially over the last four decades. Fuelled by conditioning factors such as rapid urbanisation, demographic change, large-scale migration, and travel, dengue is now endemic in most countries of the tropics, and about 925 million people now live in urban areas that are at high risk of dengue infection. More recently Zika, another mosquito-borne virus, has been implicated in the causation of microcephaly in infants, and encephalitis in adults. In conjunction with concerns about expanding mosquito habitats, and global movement of humans on significant scales, there is a compelling need to find new solutions for the prevention this family of diseases, as well as diagnoses of extent and type of exposure, as this information has significant implications for subsequent treatment and strategies for prevention. A particular challenge in this family of diseases concerns the fact that while initial exposure gives rise to mild disease, subsequent exposure to other related viral strains can result in severe illness and death, as a consequence of known mechanisms associated with the immune system, which act to make the disease symptoms considerably worse. It follows that both specific diagnosis and new vaccine designs will be required to control these diseases effectively. This project aims to exploit specific cell factories, together with our understanding of key protein structures in flaviviruses, to generate novel non–natural proteins which will have the capability both of enabling strain-specific diagnosis, as well as inducing protection in vaccination programs of carefully screened individuals, without predisposing to haemorrhagic fever (which is a recognised risk for existing vaccine designs).
Vacation Scholarship: University of Kent – Functional characterisation of a putative succinate efflux pump from Corynebacterium glutamicum.
Succinate is a key precursor in the production of biodegradable plastics and fabrics. The majority of industrially produced succinate is derived from petrochemical precursors. However, several microbial species have been engineered to maximise succinate production during fermentation. A succinate efflux pump, SucE, was recently identified in C. glutamicum, which substantially increases succinate production when overexpressed. However, the structure, mechanism, energetics and substrate specificity of this transporter remain unknown. A comprehensive understanding of SucE’s transport mechanism could allow us to manipulate this transporter and/or it’s energy source to make succinate (and possibly other dicarboxylic acid) efflux more efficient, potentially increasing the succinate yield of C. glutamicum. This project fits perfectly within the remit of CBMNet as it is centred on understanding how an industrially important chemical is transported across the bacterial membrane. The aims of this project are to; 1) clone sucE from C. glutamicum into an E. coli expression vector, 2) optimise the expression and purification conditions, 3) assay SucE function using in vivo succinate accumulation assays, and 4) reconstitute SucE into liposomes for in vitro transport assays.
Vacation Scholarship: University of Leeds – Durable vesicles for stabilisation of membrane proteins in biotechnology
Hybrid vesicles, which combine the biofunctionality of phospholipids with the stability of block copolymer membranes, can enhance the functional durability of membrane proteins. We have demonstrated that hybrid vesicles extend the functional half-life of cytochrome bo3 from 1-2 weeks in proteoliposomes to 4-6 months in hybrids (Chem. Commun. 52, 11020, 2016). The student will aim to:
1. Successfully reproduce functional reconstitution of cytochrome bo3 into hybrid vesicles (training).
2. Characterise proton pumping by cytochrome bo3 in hybrid vesicles using a pH-sensitive fluorophore.
3. Test whether (i) using a different triblock copolymer, or (ii) protein reconstitution using SMALPs has advantages over our existing materials and protocols.
4. (If time permits) co-reconstitute cytochrome bo3 with F-ATPase to create a proto-organelle to generate ATP.
Vacation Scholarship: University of Nottingham – Purification of membrane transporters to identify topology and binding sites by mass spectrometry.
Multidrug (MDR) pump show an unusually broad substrate specificity, which is poorly understood. This lack of knowledge undermines their potential use in IBBE applications where this polyspecitify may be harnessed in engineered microorganisms. Our group has an excellent track record in working with MDR pumps and now wish to harness recent advances in protein labelling and mass spectrometry (MS) to see if we can map the polyspecific binding sites of MDR pumps using MS.
Vacation Scholarship: University of Kent – Vitamin B12 into yeast.
We will determine if Saccharomyces cerevisiae can transport vitamin B12 (cobalamin) into the cell. A recent paper concerning the production of butanol isomers suggests that this is possible (see Curr Opin Biol 2015, 33: 1-7) but we would like to provide definitive evidence and quantify the levels of cobalamin that can be accumulated. We will investigate the ability of S. cerevisiae to absorb B12 using two complementary approaches. Firstly, we will grow the yeast in the presence of a range of different concentrations of vitamin B12. After growth, the cells will be thoroughly washed and then lysed. The supernatant from the lysed cells will then be applied to a very sensitive B12-dependent microbial plate assay. This will allow the student to compare B12 uptake against external concentrations of added B12. Secondly, we have made a number of B12 fluorophores, whereby we have attached a fluorophore to the B12 mainframe. These fluorescent probes are taken up by bacteria, algae and worms. With our new confocal microscope system we will be able to follow the movement and accumulation of the fluorophore within the S. cerevisiae also.