Preparation Of Foldase DsbA And Its Application In Assisting Protein Refolding In Vitro Using Statistical Experimental Design

In recent decades, the gordian knot in genetically engineered proteins industry is how to efficiently refold the proteins expressed as inclusion bodies. Especially, the renaturation of recombinant proteins with rich disulfide bonds is becoming a popular hotspot for many researchers. DsbA (disulfide bond formation protein A) located in the periplasm of E. coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this thesis, after recombinant DsbA was fermented and purified, DsbA-mediated in vitro refolding of model protein lysozyme was investigated. Furthermore, the heat denaturation of lysozyme was calculated by molecular simulation technique under high temperature both in water sphere and 8 M urea solution.Firstly, four most important factors affecting the DsbA expression were screened out among 17 parameters by Plackett-Burman design, which are concentration of NH4CI in the medium, final concentration of IPTG, cultivation temperature and cultivation time after induction. Hybrid design and nonlinear constrained optimization method were applied to figure out the optimal settings of fermentation procedure. After the optimization, about 51.9% higher expression compared with the basic condition was obtained at 390.4±1.8 mg per liter broth. Then purification process parameters of pH, eluting salt concentration, initial salt concentration in equilibrium buffer and loading amount were optimized by Box-Behnken design and multiple objective programming. With optimal settings, protein recovery of about 96.8% and purity of more than 95% DsbA at a concentration of 962.2μg/ml were achieved, and the whole productivity could be 377.9±1.7 mg soluble DsbA protein per liter broth.Secondly, the concentration of initial lysozyme, urea, GSSG and KCl were selected out by Plackett-Burman design in free DsbA-mediated in vitro refolding of lysozyme. Small central composite design and multiple objective programming showed that the optimal protein recovery was 99.7% and specific activity 25600 U/mg. The kinetics model of in vitro refolding of lysozyme was established with different concentration of urea, KCl, GSSG and initial lysozyme and molar ratio of DsbA to lysozyme. The kinetic constants κ2,κ3 and κ2/κ3 were calculated and the experiment data fit the models very well.Thirdly, purified DsbA was immobilized onto NHS-activated Sepharose Fast Flow gel with the capacity of about 5.3 mg DsbA/ml swollen gel. The initial concentration of lysozyme, temperature, pH, the concentration of urea and KCl and redox environment were investigated in immobilized DsbA-mediated batchwise refolding compared with dilution refolding. It was easy to recovery and separate the immobilized DsbA protein after refolding, and 82.5% activity recovery of lysozyme was still maintained after 3-run reusage, but a sharp drop appeared after that.Fourthly,5 ml swollen gel with immobilized DsbA was packed into a column to implement on-colum refolding. Under the optimal condition, the activity recovery of refolded lysozyme by on-column refolding chromatography reached about 95.7% and the refolded lysozyme with a concentration of 369μg/ml in 4.2 ml was fractionated with a loading amount of 1.6 mg denatured lysozyme. The fourth reusage still showed high performance in activity recovery, while the fifth reusage showed only 78% compared with the first run. Based upon that,1 ml of Sephadex G-100 gel functioning as the size exclusion chromatography was sequentially packed onto the top of the immobilized DsbA column, namely a conjoint column was constructed. The activity recovery of refolded lysozyme by the novel conjoint refolding chromatography reached about 92.7% and the refolded lysozyme with a concentration of 542μg/ml in 4.2 ml was fractionated, in which a loading amount was increased to 2.4 mg denatured lysozyme under the optimal condition. The conjoint column was repeatedly used at least 10 batches with the loading amount of 2.8 mg denatured lysozyme in 0.5 ml, and a higher throughput was achieved. No significant drop in activity recovery of refolded lysozyme was observed in the first six usages. Even after ten run, refolded lysozyme still could gain 57.7% activity recovery, about 70% compared with the first.Finally, the heat denaturation of lysozyme was calculated by molecular simulation at 500 K,700 K and 900 K. Three important parameters including root mean square deviation of the atoms, number of hydrogen bonds and the molecular radius of gyration implied the fully unfolding of lysozyme at high temperature. Compared with water sphere environment, lysozyme was more unfolded in 8 M urea solution at 700 K.In summary, the fermentation, purification and application of DsbA in in vitro lysozyme refolding were carried out. Important information of refolding of lysozyme with four pairs of disulfide bonds was obtained and the refolding kinetics model was established, which would be useful for foldase-mediated in vitro refolding process.