Document Type : Original Article (s)
PhD in Genetics, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
Associate Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Associate Professor, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
Associate Professor, Department of Genetics, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran
Background: In asthma, the relationship between number of eosinophils and severity of this disease supports the hypothesis that eosinophil is the major effector cell in inflammation of airway. Evolution of eosinophils is regulated by interlukin-5 (IL-5). Therefore, by blocking IL-5, at least one major reason of asthma would be prevented. To produce antagonists against IL-5 (like aptamer), it is necessary to have this protein in large scale and high purity. This study aimed to optimize IL-5 protein expression of BL21 strain of Escherichia coli (E. coli) to be used instead of antibody.Methods: At first, complementary DNA (cDNA) construct encoding IL-5 was designed, and was ordered to be produced in pET28a vector. Expression vector was transformed into competent E. coli Bl21 (DE3) origami. Then, protein expression was optimized by altering temperature, incubation time, and the amount of isopropyl β-d-1-thiogalactopyranoside (IPTG). Protein expression was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot in different levels of the test.Findings: The optimum conditions for protein expression were gained when the density of bacteria at the OD600 reached to 0.6 to 0.8, and culturing was done at 29 °C for 18 hours and 150 rpm, andinduction with 1mM IPTG. There was a 13-kDa protein band on SDS-PAGE and western blot that confirmed the expression of IL-5 protein.Conclusion: This protein can be used for producing aptamers against IL-5 and enzyme-linked immunosorbent assay (ELISA) kit for measuring IL-5. In all these process, there is no need to perfect folding of the protein. Therefore, the expression can be done in prokaryotic system, as it has high efficiency.