Understanding the rules of life

Category: CASE Studentships

Structure-function analysis of synthetic vesicle formation

Primary Supervisor

Professor Syma Khalid – University of Southampton

Co-Supervisor(s)

Professor Dan Mulvihill – University of Kent

Dr Chris Lennon – Fujifilm-Diosynth

Summary

The isolation of recombinant proteins from large scale fermentation cultures is a major challenge to researchers.

The ability to target the secretion of recombinant proteins into culture media is attractive as it improves efficiency and reduces costs, as well as often improving protein solubility. Currently there are no established methods for transporting recombinant proteins across bacterial membranes, or for generating vesicles to facilitate the subsequent isolation and purification of the recombinant protein from the culture media. multiple science images
E. coli is an attractive system for recombinant protein production in the biological research setting. It is not only cheap and easy to culture in batches to high densities, a wide range of strains, reagents, promoters and tools have been developed to facilitate the production of functional proteins in E. coli. The application of synthetic biology strategies are now overcoming limitations commonly associated with the application of post-translational modifications and correct folding of complex proteins.9 As for all expression systems, there is a need to improve protein yield and improve the efficiency of protein purification. The Mulvihill group have developed a novel system that utilises a Vesicle Nucleating polypeptide (VNp) that when expressed in the gram-negative bacterium, E. coli, drives the formation of large extracellular vesicles (Fig 1), into which recombinant proteins of interest can be targeted.

The project:

Southampton – The student will be trained in the use of atomistic and coarse-grain modelling, and use these skills to model how the VNp interacts with the bacterial inner membrane, and nucleates its deformation to drive vesicle formation. The coarse-grain models will enable the study of large membrane systems with thousands of lipids and many proteins. These simulations will provide insights into the mechanistic aspects of vesicle production. For insights into the specific atomic interactions that drive these mechanisms smaller portions of the systems will be reverse-mapped to atomistic resolution for fine grained simulations. The student will use this information to generate and model new iterations of the VNp to test predictions on key molecular interactions.
Kent – The student will clone and test the ability of these modelled peptides to drive vesicle production in E. coli. They will be trained in molecular cell biology, biochemical and live cell imaging techniques to follow vesicle formation (Fig. 1). In this way the student will iteratively refine models and experiments throughout the project to enable them to develop a detailed understanding into protein-membrane interactions.
Fujifilm – The ability of lead vesicle nucleating peptides to incorporate target proteins into vesicles will be tested within batch cultures at the Fujifilm-Diosynth site, where the student will gain hands on experience of biotechnology processes within an industrial setting.