Study Of Recombinant Construction And Expression Of Fibroblast Growth Factor21and Fusion Protein E4F21

Objective:To effectively express and prepare human Fibroblast Growth Factor21solublely in E.coli and on which sutdying the method of expressing the recombinant fusion protein E4F21is based.Methods:The DNA Sequence of the FGF21was synthetsised.The recombinant vector pET-3c-FGF-21was constructed and transformated into BL21(DE3) Star PlysS. The method of solublely expressing the recombinant FGF-21Was determined through longer expression time under37℃、32℃、23℃.The physical and chemical property of the recombinant FGF-21was determined after purification through chromatographic column.The activity of the recombinant human FGF-21to advance the absorption of Glucose was certificated on3T3-L1in vitro.The cDNA of fusion protein E4F21was synthetsised by PCR, then construced pET-3c-E4F21、pET-32a-EK-E4F21which were transformated into BL21(DE3) Star PlysS and W3110respectively. Subsequently,the method to express fusion protein was studied through altering induction time, induction Temperature and nutrient medium.Results:The method of solublely expressing the recombinant fusion protein FGF-21in E.coli was optimized as induction for12hours under23℃.The SDS-PAGE showed the percentage of the target protein was pproximately20%in recombinant E.coli and the target protein was mainly soluble.The MS-LC showed the Molecular Weight of the target protein was19539Da,Which was identical to be the form of Met-FGF-21. The recombinant FGF21obviously advanced the absorption of Glucose in vitro.Different recombinant vector pET-3c-E4F21、pET-32a-EK-E4F21were successfully constructed. However,the fusion protein E4F21was failed to be expressed throuth altering induction time, induction Temperature,nutrient medium and exprssion system. Moreover,death of bacteria after induction was found in the pressence of glucose in medium.Conclution:The recombinant human FGF-21could be solutely expressed in E.coli in high performance.A more reasonable and simple method to express the recombinant FGF-21in E.coli was provided,which lay a good foundation for future research and development of drug and was also one of guidance for the study of expression and chemical couple of fusion protein E4F21. The fusion protein E4F21could not be exprssed in E.coli.