title: [Construction, expression and preliminary pharmacokinetic analysis of IL-1ra mutants].
abstract: Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-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 Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
authors.affiliation: Institution of Biotechnology, Beijing 100071, China.
authors.forename: Yu-Xin
authors.initials: YX
authors.lastname: Wang
authors.forename: Zhi-Xin
authors.initials: ZX
authors.lastname: Yang
authors.forename: Heng-Qi
authors.initials: HQ
authors.lastname: Zhu
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Zhou
authors.forename: Pei-Tang
authors.initials: PT
authors.lastname: Huang
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000096:biosynthesis / Q000235:genetics* / Q000493:pharmacokinetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D050505:Mutant Proteins / Q000096:biosynthesis / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D050505:Mutant Proteins; D011994:Recombinant Proteins