Optimized Expression and Highly Efficient Purification of the Anti-inflammatory Drug rIL-1Ra from E. coli using Ni/Silica-Coated Magnetic Nanoparticles

Hashemzadeh, Mohammad Sadegh; Alishiri, Gholam Hossein

doi:10.30491/hpr.2025.520815.1515

Optimized Expression and Highly Efficient Purification of the Anti-inflammatory Drug rIL-1Ra from E. coli using Ni/Silica-Coated Magnetic Nanoparticles

Document Type : Original Article

Authors

Mohammad Sadegh Hashemzadeh ¹

Gholam Hossein Alishiri ²

¹ Nanobiotechnology Research Center, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

² Department of Rheumatology, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran

10.30491/hpr.2025.520815.1515

Abstract

Background: IL-1β is a key mediator of inflammation in the body. Upon inflammasome activation, the IL-1 receptor antagonist (IL-1Ra) serves as the primary natural inhibitor of IL-1β by competitively binding to its receptor, thereby limiting inflammatory signaling. Due to this mechanism, IL-1Ra has garnered significant interest as a biological anti-inflammatory drug.
Objectives: This study aimed to produce recombinant IL-1Ra (rIL-1Ra) in E. coli using optimized expression conditions and to develop a highly efficient purification process utilizing Ni/silica-coated magnetic nanoparticles.
Methods: The IL-1Ra gene was cloned into the pET-28a expression vector. The correct construction of the recombinant plasmid was verified by PCR and DNA sequencing. Expression of rIL-1Ra was carried out in E. coli BL21 (T7 Express) under optimized conditions (induction with 0.5 mM IPTG at 25 °C for 16-18 h). The expressed protein was analyzed by SDS-PAGE and Western blot. Purification was performed using Ni/silica-coated magnetic nanoparticles, followed by protein concentration via polyethylene glycol (PEG). The protein concentration was determined by Bradford assay, and the product was subsequently stabilized by buffer exchange into PBS (pH 7.4) through dialysis, supplemented with 10% glycerol, and stored at -20 °C.
Results: PCR and sequencing confirmed the successful construction of the expression cassette, showing the expected ~450 bp insert. SDS-PAGE and Western blot analyses detected a protein of approximately 19.8 kDa, confirming the expression and identity of rIL-1Ra. Maximum soluble expression was achieved under the optimized conditions. Purification using Ni/silica-coated magnetic nanoparticles yielded 10 mg of rIL-1Ra per 1000 ml of bacterial culture (10 mg/L).
Conclusion: The E. coli BL21 (T7 Express) system proved to be an effective and cost-efficient host for producing soluble rIL-1Ra. Furthermore, the use of Ni/silica-coated magnetic nanoparticles provided an efficient and scalable purification method, yielding a substantial amount of the recombinant protein suitable for further research and potential therapeutic applications.

Keywords

Interleukin 1 Receptor Antagonist Protein

Arthritis

Rheumatoid

Anti-Inflammatory Agents

Magnetite Nanoparticles

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