Novel methods for influenza drug research and development: Cloning, expression and purification of influenza neuraminidase for inhibitory assays
Influenza is a highly contagious virus which can cause mild to severe disease. The most effective drugs for treating influenza, are the neuraminidase inhibitors (NAIs), but resistance to these inhibitors has emerged. New methods to help make the process of identifying NAIs, and confirming their effectiveness, are presented in this thesis. A new method for producing NA in a mammalian cell line, HEK-293E cells, has been used to generate a crystal structure of the 1918 Spanish influenza virus. This method of NA production was enhanced by including a secretion signal from a human chemokine binding protein. The NA produced was soluble, formed the native quaternary structure and was glycosylated at three asparagine residues. This method of production of NA will be useful in further studies, such as the characterisation of NAI binding. NA inhibition assays sometimes produce false positive results, caused by quenching in a fluorometric assay. One way to correct for quenching is the use of Stern-Volmer quenching constants. In this thesis, Stern-Volmer quenching constants were determined at multiple concentrations, and a concentration dependence was observed. This refutes the current method of correction, and a new method is proposed.
Advisor: Krause, Kurt
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: influenza; neuraminidase; protein; inhibitor; crystallography; HEK cells; mammalian expression
Research Type: Thesis