| It is convinced that there exists the second genetic code, called protein folding code. How to refold inclusion bodies with high yield at high protein concentration is a challengeable problem at present. And now it is becoming one of the industrial bottlenecks of bioengineering. In order to promote this problem which be solved as early as possible, refolding of hen egg white lysozyme (HEWL) and recombinant human interferon-gamma (rhlFN-#) inclusion bodies was studied.HEWL as model protein, effect of reducing agent dithiothreitol (DTT) on refolding yield and free thiol number during denaturation and renaturation was investigated. The results showed that the optimum concentration of DTT in denaturation buffer was 30 mM while the initial protein concentration was no more than 40 mg/mL. Under this condition, there is no need to remove DTT during directly refolding by dilution. Furthermore, the condition of HEWL denaturation was studied so that the refolding yield could be improved.Protein refolding by dilution is the basis of research on other method of renaturation. According to the study on HEWL refolding by dilution at high concentration, the optimum renaturation buffer was determined. The renaturation buffer contained 3-~6 mM reduced glutathione(GSH) with 2~5 mole ratio of GSH to oxidized glutathione (GSSG) and 3-4 M Urea. The optimum concentration of urea increased slightly with the increase of denatured protein concentration. Compared with batch dilution renaturation, the final concentration of refolded HEWL was higher, and the refolding yield was increased by 10%~15% with dilution renaturation in fed-batch operation, urea concentration was decreased correspondingly. Then research with emphasis on the conditions of HEWL refolding using size-exclusion chromatography (SEC) was conducted, including column height, the amount of GSH and GSSG, the ratio of GSH to GSSG, urea concentration, flow rate, sample application(mode and volume), and protein concentration. HEWL refolding using SEC in continuous operation was firstly advised. Compared with that in batch operation, there is no evident difference in activity recovery, however the efficiency of the former was enhanced by about 25% and the renaturation buffer was also reduced.Kinetic model of protein refolding in vitro was put forward, and the model equation was sieved computionally. Then the model equation was used to stimulate theexperimental data, which were obtained from HEWL refolding by dilution. The fit coefficients were ranged from 0.98 to 0.99, which indicated that the kinetic model could well describe the process of HEWL refolding in vitro. Increasing the concentration of urea in renaturation buffer had the effects that rate constants ka and ka were decreased and the renaturation was prolonged. However, the ratio of ki to ks was increased , suggesting that urea had positive effect on inhibiting the aggregation than that on inhibiting the renaturation. It is the reason that optimum amount of urea could assist the lysozyme refolding. According to the mathematical expression of the rate constant k2, ka and the urea concentration, coupled with model equation, prediction of the refolding yield and optimization of the conditions of HEWL refolding in theory were carried out. Especially, the optimum concentration of urea can be determined in the light of the initial concentration of denatured protein, which providing important instruction for renaturation method and industrial refolding process design.With the method of single factor combining with orthonormal test, research on washing and purification of rhIFN-y inclusion bodies was proceeded. The purity of rhIFN-y in inclusion body was more than 80% with 15% loss of the target protein. Using SEC refolding rhIFN-y inclusion body, the result showed that SEC had integrative effect on not only promoting rhIFN-y refolding in vitro also purification of desired protein. Based on the method of determination of rhIFN-y activity set up in our laboratory, investigation on rhIFN-y refolding using SEC was...
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