Expression,refolding and purification of a human interleukin-17A variant |
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| Authors: | Bingyuan Wu Jennifer F Nemeth Dariusz J Janecki Brian Jones Galina Obmolova Thomas J Malia Audrey Baker Deidra Bethea M Merle Elloso Michael Naso Susann Taudte |
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| Institution: | 1. Biologics Research, Centocor Research and Development Inc., 145 King of Prussia Road, Radnor, PA 19087, United States;2. Immunology Discovery Research, Centocor Research and Development Inc., 145 King of Prussia Road, Radnor, PA 19087, United States;3. From the Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, and Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China,;4. Department of Endocrinology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China,;5. Department of General Surgery, Peking University Third Hospital, Beijing 100191, China;6. Department of Cardiometabolic Disease, Merck Research Laboratories, Merck & Co, Inc., Kenilworth, New Jersey 07033;1. Peking University Sixth Hospital, Peking University Institute of Mental Health, NHC Key Laboratory of Mental Health (Peking University), National Clinical Research Center for Mental Disorders (Peking University Sixth Hospital), Beijing 100191, China;2. National Institute on Drug Dependence and Beijing Key Laboratory of Drug Dependence, Peking University, Beijing 100191, China;3. State Key Laboratory of Natural and Biomimetic Drugs, and Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China;4. Peking-Tsinghua Center for Life Sciences and PKU-IDG/McGovern Institute for Brain Research, Academy for Advanced Interdisciplinary Studies Peking University, Beijing 100871, China;5. School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;1. Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan;2. Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan;3. Graduate School of Integrated Sciences for Life, Hiroshima University, 1-3-2 Kagamiyama, Higashi-Hiroshima City, Hiroshima 739-8511, Japan;4. Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-Ku, Sendai 980-8577, Japan;1. UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK;2. PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK;1. Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Va;2. Asthma and Allergic Diseases Center, University of Virginia School of Medicine, Charlottesville, Va;1. Universidad Autónoma de Nuevo León, Facultad de Ciencias Físico Matemáticas, División de Posgrado, Av. Universidad S/N Ciudad Universitaria San Nicolás de los Garza Nuevo León CP 66451, Mexico;2. Universidad Autónoma de Nuevo León, Facultad de Ciencias Químicas, Av. Universidad S/N Ciudad Universitaria San Nicolás de los Garza Nuevo León CP 66451, Mexico;3. Universidad Autónoma de Nuevo León, Facultad de Ingeniería Mecánica y Eléctrica, Posgrado en Ingeniería de Sistemas, Av. Universidad S/N Ciudad Universitaria San Nicolás de los Garza Nuevo León CP 66451, Mexico |
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| Abstract: | A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T7 promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris–HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2–8 °C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts. |
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