Establishment And Provement Of The Method Of Enhancing Tripartite Split-GFP By Applying GFP Binding Protein

Objective: To discover and apply GFP binding protein to research tripartite split-GFP technique based on modified GFP,superfold GFP(sf GFP)can improve this technique.Methods: 1.Select GFP:Observe fluorescence intensity after transfection to select EGFP and sf GFP.2.Establish Tripatite split-GFP Method System:Divide sf GFP into three parts,GFP1-9,GFP10 and GFP11,establish tripartite split-GFP method then.3.Selection of GBP:Survey the green fluorescence on the GFP binding protein after they function on the recombined split-GFP,then examine the advantageous GFP binding protein,observe the co-foucus of GBP and GFP,then Verify the ability of GBP on GFP recombination through in vitro experiment,find out the binding site between GBP and GFP finally.4.Protein localization ability research:Test if Tripatite split-GFP Method can really localization of protein by research on LNMB1.5.Improvement of GFP Tripatite split-GFP Method:Develope three protein into five protein and explore its practicability.Results:1.sf GFP shows stronger green fluorescent intensity through Cell transfection experiment.2.We established Tripatite split-GFP Method successfully.3.DARPin-GFP,GFP-enhancer and Nbsf GFP were selected as their different strengthening ability on recombination of split-GFP,GFP-enhancer and Nbsf GFP are stronger than DARPin-GFP.4.We tried to observe the green fluorescent on the exact position where LNMB1 should located in,the order of the fusion protein may be not reasonable,but failed.5.We failed to observe the green fluorescent in 48 hours after cell transfection,steric hindrance may be the possible reason.Conclusion:We found that sf GFP may be better for research use and selected three GFP binding protein which are capable of enhancing the renaturation and reinforce of split-GFP,also,we proved it in vitro and had carried out its improvement test.This discovery may be used for protein-protein interaction research by improve its efficiency and sensitivity.