| Liquid chromatography (LC) is one of the most effective way to separate and purify biopolymer, and also a powerful tool to be used for protein refolding. In this paper, the standard insulin from bovine pancreas was selected as the model protein to study the refolding of the reduced-denatured insulin with hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), size exclusion chromatography (SEC), respectively. The results were further tested by the separation of the PFLC fraction of reduced-denaturated insulin with reversed-phase liquid chromatography (RPLC), then detected with matrix assisted laser desorption/ ionization time of flight mass spectrometer (MALDI-TOF MS). Furthermore, the refolded insulin was also detected by ultraviolet spectrometry and fluorescence spectrometry.The principle of protein refolding and protein refolding methods were presented in the first part of this thesis. Focused on the contribution of protein folding liquid chromatography on protein folding and its development. At the end of this part, the objective and the method of this study was briefly introduced.Firstly, the refolding of reduced/denatured bovine pancreatic insulin was studied with HIC. The effects of different concentration of urea and different concentration ratio of GSH/GSSG on the forming the disulfide bonds of reduced-denatured insulin were investigated in detail. The results indicated that low concentration of urea can depress the aggregation of the reduced/denatured insulin effectively, and the glutathione redox in the mobile phase participated in the formation of the disulfide bonds, which can promote the efficiency of renaturation. At the same time, combined with mass spectrometry analysis, it was found that numbers of GSSG and GSH molecules modified with the two chains of insulin, which reduced the efficiency of renaturation of insulin. With the presence of GSH/GSSG as the ratio 6:1 in the mobile phase employed, the refolding efficiency of reduced/denatured insulin can be increased from zero to 3.5%. Furthermore, five kinds of column with different hydrophobicity were used, the hydrophobic groups were PEG-200, PEG-400, PEG-600, PEG-800, PEG-1000, respectively. The highest mass recovery of refolded insulin was obtained from the medium hydrophobicity of HIC column, that's PEG-600 HIC column. In this part, the dynamics during the reduced/denatured insulin refolding was also investigated. The results showed the effects on the refolding of reduced/denatured insulin with different residence time on the HIC column before the elution started. With the increase of residence time on the HIC column, the refolding efficiency of reduced/denatured insulin can be increased a little, but the effect was not obvious.Secondly, the refolding of reduced/denatured bovine pancreatic insulin was studied with IEC. The reduced/denatured insulin was refolded by using weak anion exchange chromatography (WAX). Effects of urea concentration, the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst on chromatographic behavior of denatured/reduced insulin on WAX were investigated in detail. The results indicated that the mobile phase with 3 mol/L urea and GSSG/Cyst as the ratio 1:6, the highest efficiency of correct refolding of insulin was obtained, the mass recovery reached 10.6%. In addition, the urea gradient on the forming the disulfide bonds of reduced-denatured insulin were investigated in detail. After optimization of the urea gradient, the mass recovery was increased to 13.3%. With the urea gradient and in the presence of GSSG/Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced-denaturated insulin can be accelerated resulting in forming the correct three disulfide bonds. The mass recovery reached 15.6%. The result was tested by the separation of the WAX fraction of reduced-denaturated insulin with RPLC, MALDI-TOF MS analysis and spectrum detection. Under this condition, it was found that there were numbers of insulin folded to a stable intermediate, which had the same molecular weight as that of native insulin but shorter retention time on the RPLC, and the similar spectral behavior. The mass recovery of this intermediate was 17.9%, and the refolding efficiency of insulin reached 33.5%.Thirdly, the refolding of reduced/denatured bovine pancreatic insulin was studied with SEC. The effects of non-oxidized mobile phase and oxidized mobile phase on the refolding of reduced/denatured insulin were studied separately and a comparison of these two methods was made. The results indicated that the disulfide bonds almost could not be formed correctly with the former condition, while under the latter condition, the mass recovery reached 4.2%. In addition, improvement of the size-exclusion chromatography refolding process was achieved by a decreasing urea gradient for the refolding of bovine insulin in this paper. Although the gradual removal of denaturant in gel filtration may also inhibit the aggregation, but because of the molecular modification, the refolding efficiency of reduced/denatured insulin was not enhanced apparently by this way. At the end of this part, Effects of the above three different protein folding liquid chromatography on the reduced/denatured insulin refolding were summarized and compared. The best result was obtained by using IEC, but the worst by using HIC. The reason for the inefficient refolding of the reduced/denatured insulin was discussed in detail.In this thesis, the refolding of reduced/denatured insulin was studied by using HIC, IEC, SEC, separately. The influence of urea concentration, mobile phase, the oxidant and reductant groups, dynamics, urea gradients and other factors on the refolding of reduced/denatured insulin were investigated. The protein refolding efficiency of reduced/denatured insulin can be increased from 2.6% to 33.5% by using weak ion-exchange chromatography. A new detection method for refolding of reduced/denatured double chains proteins was established, especially for those proteins the detection of biological activity are limited. By the detection of molecular weight with MALDI-TOF MS, the truth whether the disulfide bonds between the protein chains connected or not could be determined. Then combined with the spectral detection, the difference between the refolding protein and its native state could be obtained. In this way, the estimation whether the protein was folded back to its natural state conformation or not could be obtained easily and accurately. |
PDF Full Text Request