Abstract
Interleukin-1 beta (IL-1β) acts a precursor in the regulation of inflammatory and immune responses. Here, we describe the character and bioactivity on a circular permutation of chicken interleukin-1 beta, CP36. Circular permutation is a bioengineering method that changes the protein’s amino acid sequence but allows the protein to retain its original bioactivity, usually due to the conserved tertiary structure. In this study, circular dichroism showed that spectrum of CP36 has no difference when the protein is heated to 55℃, but the spectrum signal of wild-type chicken IL-1β decreased when the protein is heated to 55℃. These results indicated CP36 is more stable than WT chicken IL-1β. As observed in thermal resistance, result of CD spectrum showed the greater tolerance of CP36 in chemical resistance. On the functional assay of plasma cortisol level, according to the thermal stability, CP36 is more stable than WT when protein heats to 50℃. We injected heat-treatment protein, cortisol level showing CP36 retains about 84% bioactivity even heats to 65℃, while WT chicken IL-1β decreased when the temperature heats to 45-50℃, demonstrated CP36 is more stable than WT chicken IL-1β. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally inhibitor of interleukin-1 (IL-1) to compete interleukin-1 receptor (IL-1R). According to the effetely IL-1Ra competing with IL-1 to reduce the immune response, the treatment of IL-1Ra has been used on anti-inflammatory therapy on human. Here, we confirm the in vivo bioactivity and determine the characters on secondary and tertiary structure of IL-1Ra. In this study, the result of circular dichroism far-UV spectrum indicated chicken IL-1Ra is a β-trefoil protein. Crystal structure indicated chicken IL-1Ra contains 4 α-helixes and 12 β-strands. These results show that chicken IL-1Ra and human IL-1Ra are identical in structure, speculating chicken IL-1Ra has the ability to compete with IL-1. Protein was injected into wing vein to investigate whether IL-1Ra compete with IL-1. Results showed the plasma cortisol in the level on the group injected IL-1Ra with IL-1β decreased than that only IL-1β injected lasted two hours, confirming that chicken IL-1Ra has the ability to compete with IL-1β. The production of cortisol produced by adrenal gland decreased enough to inhibit the immune response stimulated by IL-1β. We also found the receptor binding residues in human IL-1Ra conserved in chicken IL-1Ra, there is only one different of five, suggesting the binding residues is conserved even in other species.