Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Iran J Immunol. 2021 Jun;18(2):141-149. doi: 10.22034/iji.2021.89103.1929.
Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA-approved biologic drug for antagonizing IL-1 in patients with Rheumatoid arthritis. The less expensive production of this drug might help reduce the final therapeutic costs.
To evaluate the possibility of producing biologically active recombinant IL-1Ra by a single-step purification procedure mediated by a self-cleavable intein.
Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) infusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations was performed on A375 and HEK293 cells treated by a constant concentration of IL-1β (2 ng/mL).
IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1β was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which was 96% for the inhibitory effects of the standard drug.
In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable other biological products.
阿那白滞素（Kineret®），一种白介素-1 受体拮抗剂，是 FDA 批准的第一种用于拮抗类风湿关节炎患者白介素-1 的生物药物。这种药物的生产成本较低，可能有助于降低最终的治疗成本。
通过一步纯化程序，评估由自切割内含肽介导的具有生物活性的重组 IL-1Ra 的生产可能性。
将 rIL-1Ra 可溶性表达于 E. coli BL21（DE3）中，通过 pTWIN-1 载体的 intein1 进行表达，并在室温下使用洗脱缓冲液（pH6.8）诱导其切割。在恒定浓度的白介素-1β（2ng/mL）处理的 A375 和 HEK293 细胞中，评估该蛋白在不同浓度下的拮抗功效。
IPTG 诱导转化有重组 pTWIN-1 的 E. coli BL21（DE3），在 SDS-PAGE 中显示出一条约 45kDa 的带，该带与 intein1-rIL-1Ra 融合蛋白有关。此外，通过观察 18kDa 处的带证实了蛋白纯化。最后，rIL-1Ra 和 Kineret®对 IL-1β 的抑制作用在对 IL-1 有反应的 A375 细胞中没有统计学意义。用 15µg/mL 的 rIL-1Ra 处理细胞时，抑制率为 86%，而标准药物的抑制率为 96%。
在这项研究中，通过一步法成功地以高纯度生产出具有生物活性的可溶性 rIL1-Ra。这种方法可以降低该蛋白的生产成本和生产时间，并且可能适用于其他生物制品。
