Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 17 2 2025 04 21 Optimizing purification and refolding conditions for the recombinant EcoRI restriction endonuclease protein in E. coli Optimizing purification and refolding conditions for the recombinant EcoRI restriction endonuclease protein in E. coli 107 132 4820 10.22103/jab.2025.24329.1626 FA Camellia Katalani Assistant Professor, Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran. Elham Soleimani Ph.D. Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran. Ghorbanali Nematzadeh Professor, Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran. Journal Article 2024 11 06 Objective: Type II restriction endonucleases are the most common type of restriction enzymes, widely used in genetic engineering, particularly in gene cloning. Among these enzymes, the EcoRI enzyme is one of the most frequently utilized in the molecular field. The conventional method for producing these enzymes involves using genetically modified strains of Escherichia coli, which have been mutated to overproduce the EcoRI enzyme. However, the purification process for these enzymes is both costly and time-consuming, as it requires multiple chromatography columns. Given that the EcoRI enzyme has a simple structure without disulfide bridges and exists as a monomer, this study investigates the recombinant production, optimized purification, and refolding of this enzyme.<br />Material and methods: The EcoRI gene was isolated from the bacterium Escherichia coli (E. coli RY13) and cloned into the expression vector pET28 for recombinant expression in E. coli BL21 (DE3). The optimal conditions for protein expression were investigated, including the type of inducer,its concentration, and the temperature after induction. Protein expression was evaluated in terms of soluble protein or inclusion bodies using SDS-PAGE gel analysis. The recombinant protein was then purified and refolded using affinity chromatography and dialysis methods. Finally, the enzyme activity was compared to that of the commercial EcoRI enzyme from Thermo<br />Results: The optimal conditions for the expression of the EcoRI were determined to be 0.8 mM IPTG concentration and a temperature of 28°C. The results of the analysis of the recombinant protein expressed on SDS-PAGE gel indicated that the EcoRI was expressed as inclusion bodies. The solubilization of these inclusion bodies under mild conditions and refolding with 3 M urea demonstrated that the inclusion bodies were of a non-classical type. Furthermore, results from the refolding process, using both dialysis and Ni-NTA column, showed that the yield of refolding on the resin was higher than that obtained through dialysis. Enzymatic digestion reactions using the produced enzyme and a commercial enzyme indicated that the purified recombinant enzyme was capable of cutting plasmid DNA, similar to the commercial enzyme.<br />Conclusion: Refolding of inclusion bodies on the column resulted in significant time and cost savings, demonstrating greater efficiency compared to the dialysis method. Objective: Type II restriction endonucleases are the most common type of restriction enzymes, widely used in genetic engineering, particularly in gene cloning. Among these enzymes, the EcoRI enzyme is one of the most frequently utilized in the molecular field. The conventional method for producing these enzymes involves using genetically modified strains of Escherichia coli, which have been mutated to overproduce the EcoRI enzyme. However, the purification process for these enzymes is both costly and time-consuming, as it requires multiple chromatography columns. Given that the EcoRI enzyme has a simple structure without disulfide bridges and exists as a monomer, this study investigates the recombinant production, optimized purification, and refolding of this enzyme.<br />Material and methods: The EcoRI gene was isolated from the bacterium Escherichia coli (E. coli RY13) and cloned into the expression vector pET28 for recombinant expression in E. coli BL21 (DE3). The optimal conditions for protein expression were investigated, including the type of inducer,its concentration, and the temperature after induction. Protein expression was evaluated in terms of soluble protein or inclusion bodies using SDS-PAGE gel analysis. The recombinant protein was then purified and refolded using affinity chromatography and dialysis methods. Finally, the enzyme activity was compared to that of the commercial EcoRI enzyme from Thermo<br />Results: The optimal conditions for the expression of the EcoRI were determined to be 0.8 mM IPTG concentration and a temperature of 28°C. The results of the analysis of the recombinant protein expressed on SDS-PAGE gel indicated that the EcoRI was expressed as inclusion bodies. The solubilization of these inclusion bodies under mild conditions and refolding with 3 M urea demonstrated that the inclusion bodies were of a non-classical type. Furthermore, results from the refolding process, using both dialysis and Ni-NTA column, showed that the yield of refolding on the resin was higher than that obtained through dialysis. Enzymatic digestion reactions using the produced enzyme and a commercial enzyme indicated that the purified recombinant enzyme was capable of cutting plasmid DNA, similar to the commercial enzyme.<br />Conclusion: Refolding of inclusion bodies on the column resulted in significant time and cost savings, demonstrating greater efficiency compared to the dialysis method. https://jab.uk.ac.ir/article_4820_f538a95505cd4fc9a0c621a334bd866a.pdf