Studies Of Protein Refolding Facilitated By Folding Aids

Protein refolding is of significance not only for the industrial recovery ofrecombinant proteins expressed as inclusion bodies in microbial cells, but also for thebetter understanding of the mechanisms that lead to some serious diseases caused byprotein misfolding. In this thesis, aimed at the development of efficient methods forpreparative protein refolding, the author has, using chicken egg white lysozyme as amodel, studied protein refolding facilitated by various folding aids as well as bychromatographic methods.At first, the refolding of denatured-reduced lysozyme assisted by molecularchaperones GroEL/GroES, immobilized GroEL and immobilized GroELchromatography was investigated. It was found that there existed an optimumguanidinium chloride (GdmCl) concentration of about 1 mol/L at which the highestyield of specific activity (97%) was obtained. Using the immobilized GroEL column,a refolding productivity of 54 mg/L.h was reached at an overall yield of 81%. Thisvalue of productivity was over four times larger as that of the size exclusion refoldingchromatography reported previously, indicating the advantage of the present system atproducing a high throughput in protein refolding operations.To elucidate the kinetic behavior of lysozyme refolding assisted by the molecularchaperone GroEL, a simplified kinetic model was established based on thecompetition between protein folding and aggregation. In the presence of GroEL andATP, the aggregate formation was a second order reaction. The presence of ATP issignificant to release the folding intermediate from binding to GroEL. Thecooperation of GroEL and ATP could increase the yield to two times that of therefolding by direct dilution. Under a certain enzyme concentration, a proper ratio ofGroEL to lysozyme could reach a high refolding yield at high folding rate.Then, the artificial chaperone system, cetyltrimethylammonium bromide (CTAB)and β-cyclodextrin (β-CD), was employed to help lysozyme renaturation. It was foundthat in the presence of the artificial chaperones, the refolding yield was 70% increasedas compared to the direct dilution, and 40% increased as compared to the β-CDassisted refolding. Kinetic analysis showed that, in the presence of the artificialsystem, the aggregate formation was a third order reaction, the same as that of therefolding by direct dilution. The effects of both GdmCl and the artificial chaperoneswere to suppress the aggregation process, and the results obviously indicated thecooperative effect of GdmCl and the artificial chaperones on enhancing proteinrenaturation. By the cooperative effect, over 80% yield of lysozyme at a concentrationas high as 1 mg/mL was achieved. In addition, in the presence of the artificialchaperones, lysozyme refolding could be realized at high yield and folding rate at alow GdmCl concentration.Based on the above study of artificial chaperones, an integrative refoldingmethod of size exclusion chromatography (SEC) incorporating with the artificialchaperone system was proposed and demonstrated experimentally. By the integrativerefolding technique, higher renaturation yield was obtained at higher flow rate thanthat of SEC without the artificial chaperones. Thus, use of the SEC incorporating withthe artificial chaperone system would greatly benefit in reaching a high refoldingproductivity. Moreover, the integrative method has proved useful in continuousprotein refolding.Finally, the author has explored the effect of various simple dilution additives,such as acetamide, acetone, thiourea, L-arginine and glycerol, on protein refolding.The kinetic behavior was studied utilizing the simplified model describing thecompetition between first-order folding reaction and third-order aggregation. It wasfound that the protein folding aids could be categorized into two groups. One of themat proper concentrations, such as acetamide, acetone, thiourea and L-arginine,stabilized unfolded protein or folding intermediates, similar to GdmCl. In thepresence of these additives, both the intramolecular folding and the intermolecularinteractions were inhibited, so the folding rate decreased with increasing theirconcentration, and there was an optimum concentration to produce a high refoldingyield. The other group was protein stabilizers such as glycerol. In the presence of thiskind of folding aids, the refolding yield and rate were enhanced by increasing itsconcentration until a plateau value.