High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment |
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| Institution: | 1. Department of Biochemistry and Molecular and Cell Biology, Spain;2. Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain;3. Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain;1. Department of Forensic Medicine, College of Medicine, Seoul National University, Seoul 110-799, South Korea;2. Department of Forensic and Investigative Genetics, Institute of Applied Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA;3. Institute of Forensic Medicine, College of Medicine, Seoul National University, Seoul 110-799, South Korea;4. Department of Anatomy, College of Medicine, Seoul National University, Seoul 110-799, South Korea;1. Department of Sociology, Simmons College, Boston, Massachusetts;2. Master of Public Policy Program, Simmons College, Boston, Massachusetts;1. Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBBA-CONICET, Buenos Aires, Argentina;2. ULB-Neuroscience Institute, Universite Libre de Bruxelles, Bruxelles, Belgium;3. RNA Cell Biology Laboratory, Fundación Instituto Leloir and IIBBA-CONICET, Buenos Aires, Argentina;4. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and IIBBA-CONICET, Buenos Aires, Argentina;5. Protein Physiology Laboratory, Universidad de Buenos Aires, CONICET, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina;1. Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland;2. Department of Integrative Biology and Pharmacology, University of Texas, Health Science Center – Houston, Houston, TX 77225, USA |
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| Abstract: | Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation. |
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