Preparation Of Mini-chaperone And Its Preliminary Application In The Recombinant Protein Refolding In Vitro

The apical domain of GroEL(residues 191-345)was expressed in E.coli to give a functional mini-chaperone, and the refolding yields of scorpion toxin Cn5, Cyclophilin A and IGPS were improved remarkably assisted by mini-chaperone. In order to find its potentially broad application for more proteins, the mechanism of refolding proteins assisted by mini-chaperone need to be further investigated. In this thesis, the plasmid containing the gene of mini-chaperone was successfully transformed into E.coli, the culture condition and expression condition of the transformant was optimized and the purified recombinant mini-chaperone was applied to assist refolding proteins in vitro.Firstly, according to the nature of the vector, E.coli BL21 was chosen to be the host cell, and the transformation was proved to be a success. Based upon the relative expression amount of mini-chaperone and according to the relative expression amount of mini-chaperone, the No.l strain was selected to be the seed.Then, the optimum culture condition of mini-chaperone was studied in detail. M9 medium with carbon source and inorganic salts was found to be suitable for the expression of mini-chaperone GroEL(191-345) by E.coli. M9 medium was then reformulated and the optimum temperature, oxygen demand and induction conditions etc. were carefully investigated to improve the mini-chaperone production, the yield of mini-chaperone expressed in Kcoli BL21 reached 556.3 mg/L.For the mini-chaperone was a fusion protein with a His6-tag at N terminal, it was chosen to be purified by IMAC with the Ni-NTA column. Harvesting the cells by spinning at 6000rpm at 4癈, re-suspending the cells, cracking the cells by sonication, collecting the supernatant by spinning at lOOQOrpm, the sample was loaded for IMAC chromatography. By carefully studied the imidazole concentration in the process of adsorption and elution, pure mini-chaperone was gained by one step. The purity of mini-chaperone gained by IMAC was checked by SEC, and also the molecular weight of mini-chaperone was confirmed to be 18kDa by MALDI-TOF-MS.Finally, the application of mini-chaperone in refolding recombinant human interferon- y was studied. The main influencing factors of the refolding conditions were investigated, and the refolding yield of IFN-y could improve near 10 folds while adding the 5:1 mini-chaperone to 0.22mg/ml IFN-y in batch. That refolding yield also promised to the further application in other genetically engineered productions.