Comparative Purification Strategies for Recombinant Canine Interferon γ in Escherichia coli: Denaturing versus Native Conditions

Interferon gamma (IFNγ) is a critical cytokine that activates various immune cells, including macrophages, NK cells, T cells, and even non-immune cells, to fight particularly intracellular infections and cancers. But to use this as medicine for dogs, it is necessary to purify IFNγ for dogs due to its species-specificity. However, efficient purification protocols for recombinant canine IFNγ (rcIFNγ) from Escherichia coli that ensure protein stability remain poorly established. In this study, we systematically compared two distinct purification strategies: a denaturing method (lysis with urea) and a native method (lysis without urea), each followed by subsequent purification steps. The two strategies yielded markedly different outcomes. The denaturing protocol resulted in a highly pure and intact monomeric protein. In contrast, the native protocol led to severe proteolytic degradation of the rcIFNγ, resulting in multiple smaller fragments. This present study suggests that it is crucial to explore various conditions for developing stable purification protocols and formulations for rcIFNγ.