Document Type : Research Paper
Authors
1
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
2
1. Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran./ 2. Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
10.30498/ijb.2021.278685.3021
Abstract
Background: Interleukin-6 (IL-6) has undeniable roles in inflammatory processes due to autoimmune diseases. In this regard, soluble receptors are considered a potential approach to mitigate its inflammatory effects and modulate its physiological effects by reducing the IL-6 binding to cell surface-specific receptors.
Objective: This study aimed to produce IL-6 receptor (IL-6R) in soluble form with enhanced affinity to IL-6 without signal transduction ability.
Materials and Methods: The 3D structure of IL-6R with the selective mutations for enhancing the IL-6 binding, with minimum ability to signal transduction (mIL-6R), was predicted using Modeller 9.19. This mutated form was docked to IL-6 and gp130 (a part of the native IL-6 receptor involved in signal transduction) by the HADDOCK2.2 web server. The expression of mIL-6R was performed in E. coli BL21 (DE3), using pTWIN-1 plasmid as its linkage to the Ssp Intein. IMPACT system manual was used to purify the protein at 25 °C overnight. Next, ELISA was performed to compare the affinity of mutated and native IL-6R to IL-6. Finally, A549 cells were used to compare the inhibition of cytotoxic effects of native and mutated IL-6R.
Results: In the silico section, results established the stability of mutant’s structure with more and less affinity to IL-6 and gp130, respectively. The expression and purification results showed bands of about 50 and 23 kDa, representing the correct size of the Intein1-mIL-6R fusion protein and cleavaged mIL-6R in SDS-PAGE, respectively. Furthermore, a significant enhancement in the affinity of mutated IL-6R to IL-6 was observed compared to the native receptor. Finally, A549 cells showed more cytotoxic effects followed by treating with mutated IL-6R in comparison to cells treated with native soluble IL-6R.
Conclusion: The recombinant production of a mutated form of IL-6R with the potential ability to antagonize the IL-6 inflammatory effects confirmed with in silico studies was successfully performed for the first time to create a new drug candidate for suppressing the inflammatory effects of IL-6.
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