Abstract
Influenza is a global health concern that continues to cause considerable mortality and economic loss each year. This disease is drug-treatable; however, drug-resistant strains of the virus are emerging, emphasising the need for research into new treatments. Effective drug development relies on having a dependable, and pure source of target proteins for testing compounds against. Neuraminidase (NA), a key protein in the influenza virus life cycle and one of two FDA-approved targets for influenza is the protein being investigated in this research.
The overall aim of this research was to optimise and adapt an existing protocol for producing NA utilising Human Embryonic Kidney-293-6E cells to achieve greater yields of NA from the purification process. This aim was achieved through various means; by improving the amount of protein captured during chromatography steps; by the elimination of an unnecessary chromatography stage; and by utilising urea as a chaotropic agent to remove persistent contaminant co-purifying proteins Heat Shock Protein-70 and Calsyntenin-1.
The findings presented in this thesis form the basis of an improved purification protocol that shortens the purification workflow, increases reproducibility, and delivers highly pure, active NA, which is suitable for inhibitor screening and downstream structural analysis.