T4 bacteriophage-specific dihydrofolate reductase: purification to homogeneity by affinity chromatography |
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| Authors: | J S Erickson C K Mathews |
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| Affiliation: | 1. Structural Biology of Molecular Machines Group, Protein Structure & Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark;2. Structural Molecular Biology Group, Protein Structure & Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark;3. Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen, Denmark;1. Department of Biochemistry, University of Toronto, MaRS West Tower, 661 University Avenue, Toronto, ON M5G 1M1, Canada;2. Department of Molecular Genetics, University of Toronto, MaRS West Tower, 661 University Avenue, Toronto, ON M5G 1M1, Canada;3. Program in Molecular Structure & Function, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;4. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA;1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA;2. Center for Genomic and Computational Biology, Duke University, Durham, NC 27705, USA;3. Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA;4. Duke Institute for Brain Sciences and Duke Regeneration Center, Duke University Medical Center, Durham, NC 27710, USA;5. Departments of Cell Biology and Neurobiology, Duke University Medical Center, Durham, NC 27710, USA;1. Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan;2. Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan;3. Department of Food Science, Rutgers University, New Brunswick, NJ, USA;4. Department of Biomedical Informatics, Asia University, Taichung, Taiwan;5. Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan;6. Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan;7. Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan;1. Department of Internal Medicine IV, University Hospital Tübingen, Tübingen, Germany;2. Institute of Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich at the University of Tübingen, Tübingen, Germany;3. German Center for Diabetes Research (DZD), Tübingen, Germany;4. Section of Experimental Radiology, University Hospital Tübingen, Tübingen, Germany |
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| Abstract: | Dihydrofolate reductase specified by bacteriophage T4 has been purified 6,000-fold by a simple affinity chromatographic method employing N10-formylaminopterin (4-amino-10-formylpteroylglutamate), bound by peptide linkage to the solid support aminoethyl Bio-Gel P-150. About 4.5 mg of enzyme (80% recovery) can be obtained from 500 g of infected cells. The product is homogeneous in two different electrophoresis systems and shows a molecular weight of 29,000 as determined by sodium dodecyl sulfate gel electrophoresis. Inhibitor studies show a single binding site per enzyme molecule for folic acid analogues. |
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