Preparation Of Foldase And The Protein Refolding Assisted By DsbA In Vitro

How to refold inclusion body proteins with many pairs of disulfide bonds into natural conformation with high efficiency is a challengeable problem at present. DsbA, existing in the periplasm of E. coli, is a kind of disulfide bond isomerase, which assists the folding of the disulfide bond containing proteins to achieve biological activities. In the thesis, the plasmid containing the gene of dsbA was successfully transformed into E.coli, the culture conditions of the DsbA transformant in the fermentation and purification were optimized, then the purified DsbA was applied to assist the renaturation of lysozyme in vitro.Firstly, the plasmid pAVD63 coded with dsbA gene was tramsformed into E.coli BL21 (DE3) and the target protein was expressed intracellularly.Secondly, statistical experimental designs were used to optimize the fermentation process of recombinant DsbA in E.coli. Four most important parameters, which affect the DsbA expression, were screened among 17 parameters by Plackett-Burman design. Then hybrid design was applied to fit a response surface function, where stationary point was a saddle point with neither global maximum nor minimum. Finally, the optimal settings of fermentation parameters were calculated using the nonlinear constrained optimization method. Verification experiment showed that the settings resulted in 50.14% higher expression after the optimization.Thirdly, after the cells were collected by centrifugation and disrupted by ultrasonic with ice bath, DsbA was purified by ion exchange chromatograpy (IEC). In order to improve the efficiency and DsbA recovery of the purification process and shorten the production period, the IEC process was optimized by Box-Behnken design, the influence of pH, step of elution, initial salt concentration and loading amout were studied. The area of DsbA elution peak was set as the response to fit the response function, and three objects were integrated together by effectiveness coefficient method in MOP (Mutiple Objective Programming) and finally the optimized process was achieved: 20mL Q Sepharose Fast Flow gel packed column, pH 8.3, elution at step of 5.88% (percent of the buffer B, pH 8.3, 16.7mM Tris-HCl, 2M KCl), initial salt concentration 16.7mM, loading amount 9.25mL. The verification experiment showed the setting resulted in 4301.8 mAU·mL of the area of the DsbA elution peak, while the error was only ±2.4% compared with the predicted value. In the whole process, 377.9±1.74mg purified DsbA per liter fermentation broth was obtained, andthe protein recovery was 96.8%, the purity was more than 95%. The high purity of DsbA was identified by peptide mass finger print (PMF), and the molecular weight was ascertained to be 23kD.Finally, DsbA was applied to assist the refolding of the model protein lysozyme. Plackett-Burman design was employed to investigate 9 factors which influenced the lysozyme protein recovery and specific activity, and 5 most important factors were screened out: initial lysozyme concentration, urea concentration, KC1 concentration, GSSG concentration and the molar ratio of GSH to GSSG. Furthermore, these five significant factors were studied in details. At optimized experimental conditions, the activity recovery was increased from 44.52% to 75.85% in this DsbA mediated refolding system compared with direct dilution refolding when the initial lysozyme concentration was 250 μg/mL, This research provides an alternative method for the refolding of recombinant proteins expressed as inclusion bodies in vitro with many pairs of disulfide bonds.