Soluble expression and purification of bioactive interleukin 33 in E. coli |
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Authors: | Bich Hang Do Sangsu Park Grace G. Kwon Minh Tan Nguyen Hyo Jeong Kang Jung-A Song Jiwon Yoo Anh Ngoc Nguyen Jaepyeong Jang Mihee Jang Sunju Lee Seoungjun So Sungrak Sim Jonghwa Jin Kyung Jin Lee Mark J. Osborn Han Choe |
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Affiliation: | 1.Department of Physiology, Asan-Minnesota institute for Innovating Transplantation, Bio-Medical Institute of Technology,University of Ulsan College of Medicine, Asan Medical Center,Seoul,Korea;2.Osong Medical Innovation Foundation, New Drug Development Center,Division of Drug Screening and Evaluation,Chungbuk,Korea;3.Department of Convergence Medicine, Asan Institute for Life Sciences,University of Ulsan College of Medicine, Asan Medical Center,Seoul,Korea;4.Department of Pediatrics, Division of Blood and Marrow Transplantation, Center for Genome Engineering, Stem Cell Institute,University of Minnesota,Minneapolis,USA |
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Abstract: | Interleukin-33 (IL-33) is one of the important alarmins of the immune system and possesses dual functions as an anti- or pro-inflammatory molecule. The production of this cytokine in E. coli is hampered by the insoluble expression in the cytoplasm, resulting in inclusion body formation. In this study, the expression of IL-33 was optimized by fusing the N-terminus of IL-33 with several solubilizing tags that act as chaperones for proper protein folding: maltose binding protein (MBP), b´a´ domain of protein disulfide isomerase (PDIb´a´) and glutathione Stransferase (GST). The expression of the fusion proteins was stimulated by 0.5 mM IPTG at different temperatures, 37, 30, 25, and 18°C. As a result, IL-33 was expressed highly and in soluble form in the cytoplasm of E. coli when fused with MBP or PDIb´a´ tags in the presence of 0.5 mM IPTG at 25 or 30°C. We describe a simple purification procedure of IL-33 from the PDIb´a´-IL-33 construct using immobilized metal affinity chromatography (IMACs) with supplementary of tobacco etch virus (TEV) protease for tag removal. The high bioactivity of purified IL-33 on the proliferation and activation of macrophages was confirmed by MTT and nitrite releasing assays using RAW 264.7 These data show an improved method for producing high grade and yield IL-33. |
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