BiFC, Erythropoietin receptor, Protein engineering, Protein modelling
Table Of Contents
I. Introduction 1
II. Materials and methods 4 1. Designing biosensor system with spGFP assembly system 4 2. Transformation, culture and overexpression on e.coli system 4 3. Purification of target protein 5 4. Normalizing molar ration of sensor molecules 6 5. Detection of signal emission from biosensor system. 7 6. Cloning of target gene 7 7. Design of novel peptide ligand of erythropoietin receptor 8 8. Statistical analysis 9
III. Results 10 1. Biosensor property configuration 10 2. Biosensor activity confirmation 12 3. Evaluating activity of denatured EPO 15 4. Testing EPO’s interaction between spGFP domains 17 5. Cloning another biosensor with different receptor system 19 6. Ligand design with RFdiffusion – single binding peptides 21 7. Dimerization screening of designed ligands by BiFC – single binding peptides 23 8. Ligand design with RFdiffusion – dual binding peptides 25 9. Dimerization screening of designed ligands by BiFC – dual binding peptides 26 IV. Discussion 30 V. Conclusion 33 VI. References 35
