Elevated levels of the pro-inflammatory cytokine Interleukin 21 (IL-21) contribute to the progression of autoimmune and inflammatory diseases by binding to its receptor (IL-21R) and activating detrimental signaling pathways in immune cells. Consequently, we developed a recombinant decoy receptor protein designed to competitively inhibit IL-21 binding to its receptor. Using the extracellular domain of IL-21R as a scaffold, five key binding-site residues (33, 38, 70, 94, and 130) were mutated with OSPREY software. Computational analyses with GROMACS selected the Q33S, E38D, M70T, L94N, and M130S mutant for its superior structural stability and high binding affinity to IL-21, with binding energy calculations confirming that the mutant successfully maintains the high baseline binding affinity of the wild-type complex. The decoy receptor protein was subsequently expressed recombinantly in E. coli Rosetta-gami. More importantly, the analysis of secondary structure revealed that the protein retained its overall secondary structure after mutagenesis, with slight alterations in alpha-helical content. Finally, the calculation of the KD value using the SPR technique demonstrated that the mutant IL-21R exhibited a 1.4-fold increase in binding affinity compared to the wild-type receptor (KD=50 pM vs. 70 pM for the wild-type), supporting its potential as a competitive IL-21R decoy receptor. Therefore, this decoy receptor could be considered an effective therapeutic agent for blocking IL21 and controlling or preventing the progression of autoimmune and inflammatory diseases. Flowchart illustrating protein engineering steps, including expression, purification, and KD measurement.The flowchart details protein engineering processes. It begins with molecular structures showing mutations (Q33S, E38D, M70T, L94N, M130S) analyzed via SPRAY and Gromacs software. Next is the construction of an expression cassette (pET21a::mutant e.d.IL21R) and protein expression in Rosetta gami strain. This leads to protein purification, SDS-PAGE analysis, and secondary structure determination. Finally, a graph shows KD interaction with IL21 at 50 pM. The decoy receptor protein, the mutant e.d.IL21R, contains five point mutations: Q33S, E38D, M70T, L94N, and M130S.The recombinant mutant e.d.IL21R exhibits a secondary structure similar to that of the wild-type e.d.IL21R.The recombinant mutant e.d.IL21R demonstrated a higher binding affinity for IL-21 than the wild-type protein.A novel therapeutic approach for autoimmune disorders using the recombinant mutant e.d.IL21R is proposed. The decoy receptor protein, the mutant e.d.IL21R, contains five point mutations: Q33S, E38D, M70T, L94N, and M130S. The recombinant mutant e.d.IL21R exhibits a secondary structure similar to that of the wild-type e.d.IL21R. The recombinant mutant e.d.IL21R demonstrated a higher binding affinity for IL-21 than the wild-type protein. A novel therapeutic approach for autoimmune disorders using the recombinant mutant e.d.IL21R is proposed.
Sara Vazifeshenas, Zahra Hajihassan, Seyed Shahriar Arab et al.· Figshare· 0 citations
Elevated levels of the pro-inflammatory cytokine Interleukin 21 (IL-21) contribute to the progression of autoimmune and inflammatory diseases by binding to its receptor (IL-21R) and activating detrimental signaling pathways in immune cells. Consequently, we developed a recombinant decoy receptor protein designed to competitively inhibit IL-21 binding to its receptor. Using the extracellular domain of IL-21R as a scaffold, five key binding-site residues (33, 38, 70, 94, and 130) were mutated with OSPREY software. Computational analyses with GROMACS selected the Q33S, E38D, M70T, L94N, and M130S mutant for its superior structural stability and high binding affinity to IL-21, with binding energy calculations confirming that the mutant successfully maintains the high baseline binding affinity of the wild-type complex. The decoy receptor protein was subsequently expressed recombinantly in E. coli Rosetta-gami. More importantly, the analysis of secondary structure revealed that the protein retained its overall secondary structure after mutagenesis, with slight alterations in alpha-helical content. Finally, the calculation of the KD value using the SPR technique demonstrated that the mutant IL-21R exhibited a 1.4-fold increase in binding affinity compared to the wild-type receptor (KD=50 pM vs. 70 pM for the wild-type), supporting its potential as a competitive IL-21R decoy receptor. Therefore, this decoy receptor could be considered an effective therapeutic agent for blocking IL21 and controlling or preventing the progression of autoimmune and inflammatory diseases. Flowchart illustrating protein engineering steps, including expression, purification, and KD measurement.The flowchart details protein engineering processes. It begins with molecular structures showing mutations (Q33S, E38D, M70T, L94N, M130S) analyzed via SPRAY and Gromacs software. Next is the construction of an expression cassette (pET21a::mutant e.d.IL21R) and protein expression in Rosetta gami strain. This leads to protein purification, SDS-PAGE analysis, and secondary structure determination. Finally, a graph shows KD interaction with IL21 at 50 pM. The decoy receptor protein, the mutant e.d.IL21R, contains five point mutations: Q33S, E38D, M70T, L94N, and M130S.The recombinant mutant e.d.IL21R exhibits a secondary structure similar to that of the wild-type e.d.IL21R.The recombinant mutant e.d.IL21R demonstrated a higher binding affinity for IL-21 than the wild-type protein.A novel therapeutic approach for autoimmune disorders using the recombinant mutant e.d.IL21R is proposed. The decoy receptor protein, the mutant e.d.IL21R, contains five point mutations: Q33S, E38D, M70T, L94N, and M130S. The recombinant mutant e.d.IL21R exhibits a secondary structure similar to that of the wild-type e.d.IL21R. The recombinant mutant e.d.IL21R demonstrated a higher binding affinity for IL-21 than the wild-type protein. A novel therapeutic approach for autoimmune disorders using the recombinant mutant e.d.IL21R is proposed.
Sara Vazifeshenas, Zahra Hajihassan, Seyed Shahriar Arab et al.· Figshare· 0 citations
Elevated levels of the pro-inflammatory cytokine Interleukin 21 (IL-21) contribute to the progression of autoimmune and inflammatory diseases by binding to its receptor (IL-21R) and activating detrimental signaling pathways in immune cells. Consequently, we developed a recombinant decoy receptor protein designed to competitively inhibit IL-21 binding to its receptor. Using the extracellular domain of IL-21R as a scaffold, five key binding-site residues (33, 38, 70, 94, and 130) were mutated with OSPREY software. Computational analyses with GROMACS selected the Q33S, E38D, M70T, L94N, and M130S mutant for its superior structural stability and high binding affinity to IL-21, with binding energy calculations confirming that the mutant successfully maintains the high baseline binding affinity of the wild-type complex. The decoy receptor protein was subsequently expressed recombinantly in E. coli Rosetta-gami. More importantly, the analysis of secondary structure revealed that the protein retained its overall secondary structure after mutagenesis, with slight alterations in alpha-helical content. Finally, the calculation of the KD value using the SPR technique demonstrated that the mutant IL-21R exhibited a 1.4-fold increase in binding affinity compared to the wild-type receptor (KD=50 pM vs. 70 pM for the wild-type), supporting its potential as a competitive IL-21R decoy receptor. Therefore, this decoy receptor could be considered an effective therapeutic agent for blocking IL21 and controlling or preventing the progression of autoimmune and inflammatory diseases.
Sara Vazifeshenas, Zahra Hajihassan, Seyed Shahriar Arab et al.· Journal of Biomolecular Stru...· 0 citations
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