Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: Purification,activity, and binding properties toward multiple pesticides |
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| Authors: | Matthew J Cheesman Matthew J Traylor Margaret E Hilton Katelyn E Richards Matthew C Taylor Phillip J Daborn Robyn J Russell Elizabeth MJ Gillam John G Oakeshott |
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| Institution: | 1. CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia;2. School of Chemistry and Molecular Biology, University of Queensland, St. Lucia 4072, Australia;3. The Bio21 Institute, The University of Melbourne, Parkville, Victoria 3010, Australia;1. State Key Laboratory of Tree Genetics and Breeding, The Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China;2. Key Laboratory of Forest Ecological Environment of Ministry of Forestry, Research Institute of Forest Ecology Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China;3. Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China;4. Department of Chemistry, University of Portland, Portland, OR 97203, USA;5. CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2001, Australia;6. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;1. College of Life and Environmental Sciences, Biosciences, University of Exeter, Penryn Campus, Penryn, Cornwall, UK;2. Cesar Australia, 95 Albert St, Brunswick, Victoria, 3056, Australia;3. School of BioSciences, The University of Melbourne, Parkville, Victoria, 3010, Australia;4. CSIRO Land and Water, Floreat, Western Australia, 6014, Australia;1. Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium;2. Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1908, XH, Amsterdam, the Netherlands;3. Bayer AG, CropScience Division, 40789, Monheim, Germany;4. Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 96, 9820, Merelbeke, Belgium;1. Department of Crop Protection, Faculty of Bioscience Engineering, Coupure Links 653, Ghent University, B-9000 Ghent, Belgium;2. Faculty of Applied Biology and Biotechnology, Department of Biology, University of Crete, Vasilika Vouton, 71409 Heraklion, Greece;3. BayerCropScience AG, Research Pest Control, Alfred Nobel Str. 50, D-40789 Monheim, Germany Bayer CropScience, Monheim, Germany;4. BayerCropScience AG, Biochemistry, Alfred Nobel Str. 50, D-40789 Monheim, Germany Bayer CropScience, Monheim, Germany;1. Institute of Insect Sciences, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insect Pests, College of Agriculture and Biotechnology, Zhejiang University, 310058 Hangzhou, China;2. Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Zhejiang University, 310058 Hangzhou, China;3. Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Science, China Jiliang University, Hangzhou 310018, China;4. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo, China;1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China;2. University of Chinese Academy of Sciences, Beijing, 100049, China;3. Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China |
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| Abstract: | Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L?1 of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects. |
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