The Hitchhiker’s Guide to Protein Expression, Purification and Characterisation

Abstract

Protein crystallography, a science in its own right, has been and still is a tool vital to our understanding of the workings of life at a cellular level. Despite living in a modern era, disease is common worldwide and antibiotic resistance is on the rise. It is therefore vital to use all tools at our disposal to tackle the problems the future holds. The aims of this thesis were to explore the key experimental stages preceding protein crystallisation, develop new ways in which we can characterise protein samples and to expand current knowledge of potential drug targets in a variety of pathogenic organisms, harmful to both humans and animals. A novel series of assay kits, namely the Durham Screens, were developed to assess protein thermal stability in a variety of solute conditions, with the potential outcome of improving protein purification and crystallisation. Furthermore, the expression purification and characterisation of potential drug targets in both eukaryotic apicomplexan parasites and pathogenic gram-negative bacteria has been explored and developed. A wide variety of protein samples have been tested using the Durham Screens and an optimised protocol proposed. Apicomplexan serine palmitoyltransferase, from a variety of species, has been successfully cloned into expression vectors and both small- and large-scale expression and purification experiments, followed by biophysical characterisation, has been completed. A bioreactor-scale expression system was developed for a potential bacterial drug target, along with an exploration into possible purification strategies. Overall, the data presented in this thesis endorses the Durham Screens as an extremely useful tool in the biophysical characterisation of protein samples in a pre-crystallisation context. Apicomplexan serine palmitoyltransferase has been validated as a potential therapeutic drug target, with molecular features distinct from its human protein counterpart. The expression and purification of Bordetella pertussis RisA has been optimised, ready for future characterisation experiments. Future work includes the potential development of a fourth thermal stability screen, optimising the expression of apicomplexan serine palmitoyltransferase and further characterising RisA to strengthen understanding of Bordetella pertussis virulence regulation.