| Liquid chromatography (LC) is one of the most effective way to separate and purify biopolymer, and is also a powerful tool to be used for protein refolding. This thesis includes two parts: Firstly the purification and separation of cytochrome-c (Cyt-C) from pig heart with simple type hydrophobic interaction chromatography (HIC) column was investigated, and the purity and mass recovery of Cyt-C can be obtained to be 99% and 98.5%, respectively. Secondly the refolding of reduced-denatured insulin from bovine pancreas with size-exclusion chromatography (SEC), HIC, and ion-exchange chromatography (IEC) was also investigated, respectively. This thesis includes five chapters as follows:1.Review: The fundamental theories of LC - stoichiometric displacement theory for retention (SDT-R) and short column theory were briefly introduced, and a new type of column -simple-type column which was designed in our laboratory based on the two theories described above was also introduced in this chapter. Then some of the basic theories and methods of protein refolding were presented and compared, and the significance of the refolding of insulin was also illuminated.2. Purification and separation of Cyt-C from pig heart by HIC. A fast method for separation and purification of Cyt-C of high purity from pig heart is presented. A simple-type column of hydrophobic interaction chromatography packed with big particles was employed for the fast purification. The purity of the purified Cyt-C was tested by reversed phase liquid chromatography (RPLC), SDS-PAGE, MALDI-TOF MS, UV spectrum, and iron content in it. Compared with the common method, the new method has advantages of spending short time, having simpler purification processes, and high mass recoveries of 98.5%, high purity of 99%. In addition, the enlarging of the column and scale was investigated primarily. The simple column used has the advantages of low prices, good separate efficiency, long-time life etc., which can be applied broadly.3. Investigation on the refolding of the reduced-denatured insulin with SEC.The reduced-denatured insulin from bovine pancreas denatured with 7.0 mol.L-1 guanidine hydrochloride (GuHCl) and 8.0 mol.L-1 urea, respectively, was refolded by using SEC. The effects of different mobile phases and concentration of urea on chromatographic behavior of reduced-denatured insulin with SEC were investigated. The results indicated that the mobile phase containing GSSG/GSH and 2.0 mol.L-1 urea was the best condition to refold insulin with SEC. These results were tested with RPLC further. In addition, the urea gradient SEC was used to refold the reduced-denatured insulin. The results showed that urea gradient SEC was also a good way to enhance the refolding efficiency of reduced -denatured insulin. Comparing the refolding efficiency of reduced-denatured insulin with dilution method, SEC and urea gradient SEC and testing the results with RPLC, it was found that SEC for protein refolding is much more advantageous than dilution method, and urea gradient SEC can be used to improve refolding efficiency of insulin more than common method. The refolding efficiency of urea reduced-denatured insulin refolded with SEC is more than GuHCl reduced-denatured insulin.4. Investigation on the refolding of the reduced-denatured insulin with HIC. The reduced-denatured insulin denatured with 7.0 mol.L-1 GuHCl and 8.0 mol.L-1 urea, respectively, was refolded by using HIC. The effects of different concentration of GSH/GSSG and urea on chromatographic behavior of reduced -denatured insulin with HIC were investigated. The results indicated that the mobile phase with GSSG/GSH of 6:1 and 2.0mol.L urea was the best condition to refold insulin with HIC, and with the increasing of the concentration of urea in the mobile phase, the retention time of the proteins was decreased. With the comparison of the refolding efficiency of insulin refolded by the dilution method and HIC, it was found that HIC is much more advantageous to be used for refolding the reduced-den... |
PDF Full Text Request