A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library |
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| Affiliation: | 1. Key Laboratory of Industrial Biocatalysis, Ministry of Education, China;2. Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;2. Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China;3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;4. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu 214122, China;1. Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom;2. Department of Life Sciences, Imperial College London, London, United Kingdom;3. Institute of Food, Nutrition and Health, ETH Zürich, Zürich, Switzerland;4. Department of Life Sciences, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom;5. London Biofoundry, Imperial College Translation and Innovation Hub, White City Campus, London, United Kingdom;6. Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London, United Kingdom;7. UK Dementia Research Institute Centre for Care Research and Technology, Imperial College London, London, United Kingdom;1. Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan;2. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan;1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;2. Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;3. School of Biotechnology, Jiangnan University, Wuxi 214122, China;1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China;2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;3. Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi 214122, China |
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| Abstract: | Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes. |
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| Keywords: | Acid phosphatase-green fluorescent protein Enzyme activity Fusion protein Linker flexibility Linker library |
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