A novel strategy to produce high level and high purity of bioactive IL15 fusion proteins from mammalian cells

In this study, we report that Lysine 86 in IL15 is responsible for the instability in mammalian cells when its C-terminus is fused to the albumin binding scFv (IL15-A10m3). We demonstrate that K86A or K86R mutants increased the expression of the fusion protein from HEK293 cells. When the wild type IL15 is used for the fusion, no recombinant IL15 fusion was detected in the culture media. Additionally, we determined that the residue 112 in IL15 is critical for the bioactivity of IL15-A10m3. Examination of single and double mutants provides a better understanding of how IL15 engages with its receptor complex to achieve full signaling capacity. The results of our experiments were successfully applied to scale up production to levels up to 50 mg/L and >10 mg/L of >95% pure monomeric recombinant fusion proteins after a 2-step purification from culture media. More importantly, the recombinant fusion protein produced is fully active in stimulating T cell proliferation, when compared to the recombinant wild type IL15.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research