background: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae.
results: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53?mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis reve... More
background: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae.
results: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53?mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17?kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli?IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5?nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96?h) and showed higher stability than E. coli?IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2?nM).
conclusion: This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli?IL-1Ra.
