Research Of IL-1ra Mutants

Interleukin-1 receptor antagonist (IL-lra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks IL-1 mediated biological responses. Recombinant human IL-1ra(rhIL-lra, Kineret) was introduced in clinical trials involving patients with RA, Between 2001-2002, rhIL-1ra was approved by the US Food and Drug Administration and by the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10000 to 100000-fold excess amounts of IL-lra are needed to relieve disease because minimal IL-1 can induce complete biological responses, the dosage of 100-150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation.In our study, we want to prolong IL-lra half life and enhance its stability of metabolism by site-specific mutagenesis.First, IL-lra mutants were established by site-specific mutagenesis, the sites of mutagenesis include R~6K~7-AA, R~93K~94-AA and K~97R~98-AA. IL-1ra and its mutants were expressed in E.coli BL21(DE3 )using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni~2+ chelate chromatography and SephadexG75 gel filtration chromatography, and the purity being more than 90%. The res μ 1t of bioactivity assay showed that the activity of mutants is as high as wt IL-lra. We characterized the pharmacokinetic profile of IL-lra and its mutants with rabbit, the third mutant's half life is 2.26 times than wt IL-1ra.Our study has provided some approaches and experience for further research to improve the metabolism stability of IL-lra.