@article { author = {Feghhi-najafabadi, Saba and Shafiee, Fatemeh}, title = {Recombinant Production of a Mutant Form of Soluble IL-6 Receptor with Inhibitory Effects against Interleukin-6}, journal = {Iranian Journal of Biotechnology}, volume = {20}, number = {1}, pages = {98-105}, year = {2022}, publisher = {National Institute of Genetic Engineering and Biotechnology of Iran}, issn = {1728-3043}, eissn = {2322-2921}, doi = {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.}, keywords = {Autoimmune Diseases,A549,IL-6R,Impact,Recombinant Production}, url = {https://www.ijbiotech.com/article_143249.html}, eprint = {https://www.ijbiotech.com/article_143249_00ca4e970cf4270d437f655d47560116.pdf} }