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.

An efficient procedure for the selection of high affinity clones from a heptapeptide phage display library was developed. Lysozyme was used as a model protein to demonstrate the selection strategy. Effect of bovine serum albumin (BSA) concentration on screening the phage library was discussed and proper BSA concentration on plate blocking was determined. The elution procedure was improved by alternatingly eluting the bound phages with glycine-HCl buffer (pH 2.2) and high-concentration target protein solution. The modified method was compared with others including the conventional protocol, and the results confirmed that the modified procedure could yield high affinity phages that might be lost by other screening methods. Through comparison of the DNA sequences of foreign peptides of the clones showing specificity to lysozyme molecules, the HWWW motif was found to be the necessary amino acid sequence for the affinity. The electrostatic and hydrophobic interactions are considered to contribute to the affinity for the protein. Moreover, protein chromatography with the immobilized HWWWPAS on Sepharose gel indicated the strong binding affinity of the peptide for lysozyme.

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.

It has been recognized that the artificial chaperone system, cetyltrimethylammonium bromide and β-cyclodextrin, is effective for enhancing protein renaturation. In this work, we studied the effect of the artificial chaperone system and guanidinium chloride (GdmCl) on the oxidative renaturation of lysozyme at 0.21-1.05mg/mL, and a kineticmodel based on the competition between protein folding and aggregation was employed to express the renaturation process. The refolding rate constant increased, while the aggregation rate constant decreased, with increasing concentration of the artificial chaperones. With increasing GdmCl concentration (0.28-2M), both rate constants decreased, but there existed a specific GdmCl concentration that maximized the ratio of the two rate constants and thus the renaturation yield. The results obviously indicated the cooperative effect of GdmCl and the artificial chaperones on enhancing protein renaturation.

Protein renaturation is of importance in the recovery of inclusion-body protein produced by recombinant microbial cells. It has been recognized that the artificial chaperone system, Cetyltrimethylammonium bromide and β-cyclodextrin, are effective in enhancing protein renaturation. Using chicken egg white lysozyme as a model protein, this work studied protein renaturation by size exclusion chromatography (SEC) incorporating with the artificial chaperone system. At first, a cooperative effect of the artificial chaperones and guanidinium chloride (GdmCl) on the protein renaturation was confirmed, and it was concluded that the artificial chaperone system promoted the renaturation of lysozyme (1.05mg/ml) in the presence of 1mol/liter GdmCl. Using the SEC (29.5×2.6cm I.D., packed with Sephacryl S-100h gel) incorporating with the artificial chaperones, higher renaturation yield was obtained at high flow rate (0.8-2.2ml/min). In contrast, using the SEC without the artificial chaperones, very low flow rate (i.e.<0.4ml/min) should be used to receive a comparable renaturation yield. Thus, use of the SEC incorporating with the artificial chaperone system would greatly benefit in reaching a high refolding productivity.