Expression, purification and biological characterisation of IFNε
2017-03-01T03:03:31Z (GMT) by
The interferons (IFNs) are a family of pleiotropic cytokines identified in 1957 based on their antiviral activity. There are three types of IFNs of which the type I IFNs are the largest family, comprising a number of subtypes such as α, β, ε, δ and κ, which code for 17 proteins in humans. In the years since their discovery, more biological activities have been attributed to them. We now know that type I IFNs have antiviral, antiproliferative, apoptotic and a host of immunoregulatory functions. Intriguingly, even though all type I IFNs signal via the same cell surface receptor, they have been found to elicit different biological activities, suggesting that while type I IFNs share an overlapping signalling pathway, their biological roles and functions are non-redundant. In 2004, our laboratory identified a novel gene in the type I IFN locus predicted to be a type I IFN based on sequence homology and designated it IFNε. Further work performed in our laboratory subsequently identified that IFNε is produced constitutively in the reproductive tract and provides host immunity to reproductive tract infections caused by Chlamydia muridarum and Herpes Simplex Virus. As IFNε was a recently discovered type I IFN, there were no commercial reagents available for its study. Therefore the aims of my project were to express and purify recombinant murine IFNε to a level of purity suitable for definitive biological characterisation. Using an insect cell expression system, I was able to successfully express His6-IFNε however the purification protocol we developed proved unsuitable for purifying IFNε as it was found to degrade during removal of the His6 tag. We therefore immunized mice with His6-IFNε and through differential screening, identified a number of IFNε specific monoclonal antibodies. The anti-IFNε antibodies proved successful in a number of applications including western-blotting, immunohistochemistry and immunoprecipitation. Using immunoaffinity chromatography, we were subsequently able to purify tagless IFNε and characterise its biological functions. Although IFNε demonstrated classical type I IFN activities such as antiviral, antiproliferative and immunoregulatory activities, these were 100 – 1000x less than IFNα and IFNβ. Among the key findings of this thesis however was that IFNε required both type I IFN receptor subunits to induce the transcription of interferon regulated genes and therefore, this is the first account of IFNε being a bonafide type I IFN. Another important discovery made was that cells of the uterine luminal epithelium are the main producers of IFNε in mice. Although it was known that Ifne1 was highly expressed in the reproductive tract, the exact site of expression remained elusive. Overall, the work performed in this thesis resulted in the production and characterisation of highly pure recombinant mouse IFNε as well as the generation of a number of anti-IFNε monoclonal antibodies. These novel tools will enable us to study the role and function of IFN in greater detail and enhance our understanding of interferon biology and immunity.