| How to refold inclusion body proteins with many pairs of disulfide bonds into natural conformation with high efficiency is a challengeable problem at present, and it is becoming one of the industrial bottlenecks in bioengineering field. In this thesis, ribonuclease A (RNase A) was employed as a model protein to understand the formation of correct three-dimensional structure in the refolding of recombinant proteins in vitro. On this basis, the refolding mechanism in vitro was discussed.At first, RNase A was denatured by urea and dithiothreitol (DTT). Effect of strong denaturant urea and DTT on enzyme activity and free thiol number was investigated. When the initial concentration of RNase A was 10mg/mL, 8M urea and 20mM DTT can be used to make it denatured completely.Denatured RNase A was diluted in excess so that it can recover the natural conformation, 80-90% activity yield was achieved under the optimum condition, i.e. 100μg/mL denatured RNase A, pH 8.0, 2mM GSH, the ratio of GSH to GSSG from 4:1 to 2:1, 0.5-2M urea in the refolding by direct dilution. Also, the structure of RNase A along with the in vitro refolding was studied by spectrofluorimetry. Deducing from the changes of fluorescence spectrum and enzyme activity during the process, the mechanism of protein refolding can be speculated. At the beginning of renaturation, secondary structure and hydrophobic domain were formed immediately, after that tertiary structure and disulfide bonds formed so as to make active sites. From the experimental data of the renaturation of RNase A with the initial concentrations ranging from 0.1 to 2.0mg/mL, the fit coefficient of second-order aggregation model ranged 0.98-0.998. Along with the increase of protein concentration, aggregation rate also increased but the rate of correct folding decreased. The existence of low concentration of urea had stronger inhibition to aggregation than to correct refolding pathway, so that it can improve the activity recovery of renaturation.Then the fermentation and purification of recombinant DsbA protein were studied. The procedure included the transformation of plasmid, fermentation, collection of cells, disruption of cells and ion-exchange chromatography (IEC) separation. As a result of the optimization of the operation conditions of fermentation and separation, high purity of DsbA was obtained, with the final concentration of 176mg/L, the separation recovery 94.4%, the purity 95% and the production yield of 365.9±1.6mg/L fermentation broth.At last, DsbA-assisted refolding of RNase A was investigated in vitro. The results showed that DsbA not only improved the refolding rate of the peptides, but also improved the activity recovery of RNase A. The reason was that DsbA had extremely high oxidizing ability and could catalyze free cysteines to form disulfide bonds. Inaddition, DsbA also played the role of protein disulfide isomerase and chaperone so that mismatched disulfide bonds could be shuttled and aggregation rate would decrease. Fitting the experimental data with the kinetic model, the results showed that the addition of DsbA could increase the refolding kinetic constant k2, but had less effect on the aggregation kinetic constant k3, so that the ratio of k2/k3 was increased as DsbA concentration increased. As a result, DsbA helped to improve the activity recovery.
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