Establishment Of Fluorescent Protein Based AMPK-targetting Drug Screening Method

Objective:This article aims to establish an efficient fluorescent protein-based AMPK-targetting drug screening method,by exploring whether there is a direct interaction between AMPK and its substrate protein,to find a new AMPK mechanism of drug action for the clinical treatment of certain diseases Such as type II diabetes,cancer,obesity,inflammation,etc.)provides a new way of thinking,but also for drug development provides a new research direction.The preliminary application mainly determines whether the effect of the drug is based on the change of the fluorescence energy and directly tests in E.coli to overcome the shortcomings of long period and high cost of testing in cells and animals and can rapidly perform drug screening in large quantities.Methods:The first part:In this thesis,we constructed a set of prokaryotic expression plasmids carrying FRET system（YFP-CFP pair）,including six prokaryotic expression plasmids:pNYFP,pCYFP,p NCFP,pCCFP,pYFP-CFP,pCFP-YFP.YFP-CFP and CFP-YFP were designed as positive controls.YFP-SA2-CFP,YFP-CTCF-CFP and an equal amount of YFP mixed with CFP（YFP+CFP）were designed as negative controls.The positive clones verified by sequencing were transformed into Rosetta（DE3）competent cells and protein expression conditions were determined by optimizing the induction temperature,IPTG inducer concentration,and induction time.The overexpressed engineering protein was passed through Ni-NTA affinity chromatography and gel filtration chromatography to purify the target protein to a purity of over 90%.The FRET signal of the same molar concentration of purified protein was detected by microplate reader.The four engineered proteins of ECFP,EYFP,ECFP-EYFP and EYFP-ECFP were scanned at the excitation light of 430 nm and 480 nm respectively and the emission of light at 480 nm and 510 nm When the excitation light is scanned,the FRET is successfully constructed after data processing and mapping.On this basis,the FRET signal in the bacterial solution expressing the engineered protein was further detected by a microplate reader to verify the possibility of directly observing the FRET signal in the cells.The second part:The AMPK substrate protein SAMS and ECFP constitute a fusion protein（SAMS-ECFP）;AMPK protein is then mutated into pharmacologically active AMPK _T172E and AMPK _T172D,and purified by Ni column and molecular sieve respectively.Preparation,fluorescence polarization and Native electrophoresis detection methods were used to study the binding of SAMS-ECFP to AMPK _T172E,AMPK _T172D,and AMPK _WT.Simultaneously,FITC-（Acp）-SAMS polypeptide was synthesized,combined with its binding to AMPK to verify AMPK and The combination of SAMS-ECFP and the activity of various proteins of AMPK purified in this study;it was also applied in the study of SA2,CTCF protein.Later use of microplate reader will study the related proteins combined with AMPK and related drug molecules for AMPK screening.Results:The first part:1.Through sequencing validation,six prokaryotic expression plasmids were constructed successfully.2.The suitable conditions for inducing the expression of the target protein were as follows:bacterial liquid turbidity OD 0.6（600 nm）,IPTG concentration 0.1 mM,temperature 16 degrees Celsius 200 rpm,overnight expression.3.The target protein can be initially separated by Ni column,and further purified by molecular sieve chromatography.4.Microplate reader results showed that clear FRET signal was generated in both in vitro purified protein and bacterial culture.The prokaryotic expression plasmids of FRET system were successfully constructed,which not only verified the feasibility under the condition of protein,but also verified the feasibility under the condition of bacterial liquid.In order to study the protein-protein interaction under live cell conditions in real time,Provide a very convenient experimental method.The second part:In the course of the experiment,a large number of related proteins required for the experiment were prepared by enzyme digestion and mutation,and they were optimized for different degrees of construction.The interaction between AMPK and its substrate protein SAMS and SA2 were studied.The CTCF protein has played a role in the research.The binding of AMPK and FITC-（Acp）-SAMS polypeptide was verified,and it was verified that the purified AMPK proteins constructed in this experiment had certain activity.Later in the drug screening work will make further attempts and exploration.Conclusions:1.A fluorescence resonance energy transfer system was successfully constructed and its feasibility under protein and broth conditions was verified.2.The binding of AMPK _T172E,_T172D mutant protein,AMPK _WT protein and FITC-（Acp）-SAMS polypeptide showed that the two mutant engineered proteins and AMPK wild type protein constructed in this part had physiological activity.And it was confirmed that the literature shows that SAMS is a direct acting substrate of AMPK.