PMID- 35811828 OWN - NLM STAT- MEDLINE DCOM- 20230214 LR - 20230214 IS - 2167-8359 (Print) IS - 2167-8359 (Electronic) IS - 2167-8359 (Linking) VI - 10 DP - 2022 TI - Surface cysteine to serine substitutions in IL-18 reduce aggregation and enhance activity. PG - e13626 LID - 10.7717/peerj.13626 [doi] LID - e13626 AB - BACKGROUND: Interleukin-18 (IL-18) is prone to form multimers resulting in inactive aggregates, making this cytokine unstable for clinical use. Therefore, mutations have been introduced into recombinant IL-18 to overcome this issue. METHODS: To prevent the formation of disulfide bonds between the IL-18 molecules, multiple mutations targeting surface cysteines (C38, C68, C76, and C127) were introduced into our previously modified human IL-18 double mutant E6K+T63A (IL-18 DM) by direct gene synthesis. The open reading frames of IL-18 wild-type (WT), IL-18 DM, and IL-18 multiple mutant E6K+T63A+C38S+C68S+C76S+C127S (IL-18 DM1234) were inserted in the pET28a expression vector and transformed into Escherichia coli Rosetta2 (DE3) pLysS cells for protein production. The inclusion bodies of WT and mutated IL-18 were extracted by sonication and refolded by stepwise dialysis using 8 M urea as the starting concentration. The refolded IL-18 proteins were tested for aggregation using the ProteoStat protein aggregation assay. Their activity was also investigated by treating NK-92MI cells with each IL-18 at concentrations of 75, 150, and 300 ng/ml with 0.5 ng/ml of human IL-12 and interferon-gamma (IFN-gamma) levels in the supernatant were evaluated using ELISA. The structure of modified IL-18 was visualized using molecular dynamics (MD) simulations. RESULTS: IL-18 DM1234 exhibited the lowest aggregation signal, approximately 1.79- and 1.63-fold less than that of the WT and IL-18 DM proteins. Additionally, the IFN-gamma inducing activity of IL-18 DM1234 was about 10 and 2.8 times higher than that of the WT and IL-18 DM, respectively. MD simulations revealed that binding site I of IL-18 DM1234 was altered mainly due to surface cysteine replacement with serine (C-to-S substitution). This is the first report showing that C-to-S substitutions in IL-18 improved its activity and stability, suggesting the use of this modified IL-18 for medical purposes in the future. CI - (c)2022 Saetang et al. FAU - Saetang, Jirakrit AU - Saetang J AD - International Center of Excellence in Seafood Science and Innovation, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand. AD - Department of Surgery, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand. AD - EZ-Mol-Design Laboratory, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Roongsawang, Niran AU - Roongsawang N AD - Microbial Cell Factory Research Team, Biorefinery and Bioproduct Technology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathum Thani, Thailand. FAU - Sangkhathat, Surasak AU - Sangkhathat S AD - Department of Surgery, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand. AD - Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Hat Yai, Songkhla, Thailand. AD - Translational Medicine Research Center, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Voravuthikunchai, Supayang Piyawan AU - Voravuthikunchai SP AD - Center of Antimicrobial Biomaterial Innovation-Southeast Asia and Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Sangkaew, Natnaree AU - Sangkaew N AD - Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Prompat, Napat AU - Prompat N AD - Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Srichana, Teerapol AU - Srichana T AD - Drug Delivery System Excellence Center and Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, Thailand. FAU - Tipmanee, Varomyalin AU - Tipmanee V AD - EZ-Mol-Design Laboratory, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand. AD - Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Hat Yai, Songkhla, Thailand. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20220705 PL - United States TA - PeerJ JT - PeerJ JID - 101603425 RN - K848JZ4886 (Cysteine) RN - 82115-62-6 (Interferon-gamma) RN - 0 (Interleukin-18) RN - 0 (Recombinant Proteins) RN - 452VLY9402 (Serine) RN - 0 (IL18 protein, human) MH - Humans MH - *Cysteine/genetics MH - Escherichia coli/genetics MH - Interferon-gamma/genetics MH - *Interleukin-18/genetics MH - Recombinant Proteins/genetics MH - Renal Dialysis MH - Serine/genetics PMC - PMC9266699 OTO - NOTNLM OT - Aggregation OT - Interferon-gamma OT - Interleukin-18 OT - Molecular dynamic simulation OT - Surface cysteine COIS- The authors declare there are no competing interests. EDAT- 2022/07/12 06:00 MHDA- 2022/07/12 06:01 PMCR- 2022/07/05 CRDT- 2022/07/11 03:39 PHST- 2022/03/15 00:00 [received] PHST- 2022/06/02 00:00 [accepted] PHST- 2022/07/11 03:39 [entrez] PHST- 2022/07/12 06:00 [pubmed] PHST- 2022/07/12 06:01 [medline] PHST- 2022/07/05 00:00 [pmc-release] AID - 13626 [pii] AID - 10.7717/peerj.13626 [doi] PST - epublish SO - PeerJ. 2022 Jul 5;10:e13626. doi: 10.7717/peerj.13626. eCollection 2022.