Abstract
The two antagonists for interleukin-1 (IL-1) system are IL-1α, and IL-1β. IL-1 receptor antagonist (IL-1RA) is another naturally occurring non-competitive antagonist. All three antagonists are bound to two receptors that are IL-R1 and IL-1R2. IL-1 is a well-studied pro-inflammatory cytokine that is involved in chronic inflammatory diseases such as rheumatoid arthritis, Parkinson's disease, Alzheimer's, stroke, and epilepsy. The existing literature suggests that interleukin-1 (IL-1) biological responses are impeded by interleukin-1 receptor antagonist (IL-1RA) belonging to interleukin-1 (IL-1) family and a natural IL-1 inhibitor acting as ‘receptor antagonist’. Various studies based on different clinical and experimental models suggest that IL-antagonism has been reported beneficial in neuropathalogical conditions, whereas, pro-inflammatory actions of IL-1 receptor antagonist (IL-1RA) seem detrimental. This study was performed to ameliorate the stability of IL-1RA mutants generated by mutagenesis at specific sites like R6K7-AA, R93K94-AA and K97R98-AA. Using pTIG-Trx expression system, mutants were expressed in E. coli BL21 (DE3) accompanied with the induction of (isopropyl β-D-1-thiogalactopyranoside) IPTG. Later on, the purification of recombinant proteins was brought about using SephadexG75 gel filtration chromatography and Ni+2 chelate chromatography. The bioactivity assay result exhibited IL-IRA activity as high as mutants’ activity. The characterization of pharmacokinetic profile of IL-1RA apace with its mutants presents the half-life of third mutants 2.11 times longer than that of wt IL-1RA. This experimental study will lead to novel approaches, experiences and baseline data, thus providing basis for further research to outdo and elevate the metabolic stability of IL-1RA.
Highlights
Interleukin 1 (IL-1) belongs to the family of cytokines and consists of two potent inhibitors of immune and inflammatory responses IL-1α and IL-1β
One step PCR was performed to amplify IL-1 receptor antagonist (IL-1RA) and IL-1RA-1 likewise, other mutants were amplified by two step PCR
After cell lysis by ultrasonication, SDS-PAGE analysis of supernatant and precipitate revealed that IL-1RA and its mutants were present in supernatant
Summary
Interleukin 1 (IL-1) belongs to the family of cytokines and consists of two potent inhibitors of immune and inflammatory responses IL-1α and IL-1β. Occurring IL-1 receptor antagonist (IL-1RA) that acts as the inhibitor of IL-1 responses is considered as the third member of this family. Interleukin-1 receptor antagonist (IL-1RA) belonging to superfamily interleukin-1 (IL-1) [3] possesses a high affinity for IL-1 receptor and bind to it without provoking signal transduction, serves as an endogenous inhibitor of IL-1. This interaction of interleukin-1 receptor antagonist (IL-1RA) with IL-1 receptor leads to the obstruction of activities of IL-1 [4]. The recombinant IL-1RA (rhIL-1RA, biopharmaceutical drug Kineret) was investigated in clinical trials dealing with patients infected with rheumatoid arthritis. The limitation or flaw associated with this therapy is the copious amount of dosage which is 100-150 mg/day, because this quantity is abundant enough to affect the psychological, financial and physical conditions of patients
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