Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides |
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| Institution: | 1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China;2. Institute of Biomedical Engineering, Imperial College, South Kensington, London SW7 2AY, UK;3. Graduate School, Chinese Academy of Sciences, Beijing, China;1. Dipartimento di Biologia, Università Federico II, 80126 Napoli, Italy;2. Laboratoire de Biochimie Théorique, CNRS, UPR9080, Univ Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris, France;3. Istituto di Chimica Biomolecolare-CNR, 80078 Pozzuoli, Italy;1. Moran Eye Center, University of Utah School of Medicine, 65 Mario Capecchi Drive, Salt Lake City, UT 84132, United States;2. SUNY Eye Institute and Department of Pathology & Anatomic Sciences, State University of New York, Medical Research Service Department of Veterans Affairs Medical Center, Buffalo, NY 14215, United States;1. Medical Laboratory Science, Department of Health Technology & Informatics, Hong Kong Polytechnic University, Hong Kong Special Administrative Region;2. Department of Medical Science, Tung Wah College, Hong Kong Special Administrative Region;3. College of Photonic & Electronic Engineering, Fujian Normal University, Fuzhou, PR China;4. University of Colorado Denver Cancer Center, CO, USA;5. School of Chinese Medicine, Chinese University of Hong Kong, Hong Kong Special Administrative Region;1. Department of Toxicology, Faculty of Pharmacy, Medical University of Gdansk, 107 Hallera Street, 80-416, Gdansk, Poland;2. Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Gdansk, 107 Hallera Street, 80-416, Gdansk, Poland;3. Institute of Organic Synthesis, Department of Organic Chemistry, Faculty of Chemistry, University of Gdansk, 63 Wita Stwosza Street, 80-952, Gdansk, Poland;1. Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany;2. Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany;3. Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany;4. Department of Gynecology and Obstetrics, Marienkrankenhaus, Hamburg, Germany;5. Department of Immunology and Medicine, Memorial Sloan-Kettering Cancer Center, New York, USA;6. Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany;7. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany;8. Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany;1. Institut für Virologie, Medizinische Fakultät “Carl Gustav Carus”, Technische Universität Dresden, Dresden, Germany;2. CRTD/DFG-Center for Regenerative Therapies Dresden, Cluster of Excellence, Technische Universität Dresden, Dresden, Germany |
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| Abstract: | An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool. |
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