Construction, expression and preliminary pharmacokinetic analysis of IL-1ra mutants.
- Author:
Yu-Xin WANG
1
;
Zhi-Xin YANG
;
Heng-Qi ZHU
;
Xiao-Wei ZHOU
;
Pei-Tang HUANG
Author Information
1. Institution of Biotechnology, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Escherichia coli;
genetics;
metabolism;
Female;
Humans;
Interleukin 1 Receptor Antagonist Protein;
biosynthesis;
genetics;
pharmacokinetics;
Mutagenesis, Site-Directed;
methods;
Mutant Proteins;
biosynthesis;
pharmacokinetics;
Recombinant Proteins;
biosynthesis;
genetics;
pharmacokinetics
- From:
Chinese Journal of Biotechnology
2006;22(3):472-476
- CountryChina
- Language:Chinese
-
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.
