This article reports oxidative protein refolding assisted by artificial chaperones in reverse micelles formed by nonionic surfactant of sorbitan trioleate modified with Cibacron Blue F-3GA in n-hexane. The denatured/reduced lysozymewas used as a model protein and cetyltrimethylammonium bromide (CTAB) and-cyclodextrin as the artificial chaperones. The use of the artificial chaperones has proved to increase the refolding efficiency of denatured-reduced lysozyme at the concentration range studied (3.5-5.9 mg/mL). Moreover, the artificial chaperones increased the refolding yield in a wide range of urea concentrations. However, the optimal urea concentration range was little affected by the presence of the artificial chaperones.

The refolding kinetic behavior of denatured-reduced lysozyme in the presence of folding aids (acetamide, acetone, thiourea, l-arginine or glycerol) was studied utilizing a simplified model describing the competition between first-order folding reaction and third-order aggregation. It was found that the protein folding aids could be categorized into two groups. One of them at proper concentrations, such as acetamide, acetone, thiourea and l-arginine, stabilized unfolded protein or folding intermediates. In the presence of these additives, the folding rate decreased with increasing their concentration, and there existed a concentration where the aggregation rate constant was minimized. So, there was an optimum concentration for the folding aids to produce a high yield. The other group was protein stabilizers such as glycerol. In the presence of this kind of folding aids, both the refolding rate and yield were enhanced by increasing their concentration to a proper value. Moreover, their effect on improving protein refolding was additive to those of the first group. So the cooperative application of the two kinds of folding aids could result in favorable refolding rate and yield of protein.

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7mg mL-1, respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, thereexisted an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3ml of the lysozyme (1mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54mg per l gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12mg per 1gel perh), indicating the advantage of the present system for producing a high throughput in protein refolding operations.

Lysozyme adsorption and purification by expanded bed chromatography of a customized Nd-Fe-B alloy-densified agarose (NFBA) gel modified with Cibacron Blue 3GA (CB) were investigated, and the results were compared with those obtained with CB-modified Streamline gel (CB-Streamline). The NFBA gel had a mean size of 102m and a mean density of 1.88g/ml. The breakthrough behavior of lysozyme was modeled considering the dispersion of the liquid and solid phases and the diffusive mass transport of protein to the solid phase. Using independently determined parameters, the model prediction agreed reasonably well to the experimental data. Although the two dye-ligand adsorbents showed nearly the same static capacity, the dynamic binding capacity of the CB-NFBA gel was nearly twice that of the CB-Streamline gel. Moreover, lysozyme was purified from chicken egg white solution by the expanded beds with the two adsorbents, and the expanded bed with the CB-NFBA gel produced much larger purification factor than that with the CB-Streamline gel.All the results indicated that the small-sized dense medium CB-NFBA gel was more efficient as an expanded bed adsorbent.