| As an important method of separation and purification of biological macromolecules, high performance liquid chromatography (HPLC) wes applied for protein refolding as an special, interesting and charming method, and has been developed into protein folding liquid chromatrgraphy (FPLC). Many kinds of standard proteins and recombinant protein drugs expressed by E. coli. as inclusion body were refolded successful by PFLC. In FPLC both the stationary phase and mobile phase can contribute to the protein refolding cooperatively, but the stationary phase played an importat role to protein refolding in PFLC. Recently, one new kind of dual-functional separation media was synthesized to be used for the protein separation not only with ion exchange chromatography (IEC) mode, but also with hydrophobic interaction chromatography (HIC) mode called 2D column. As a result, only a single column can be used to complete HIC-IEC or IEC-HIC two-dimensional liquid chromatography (2D-LC). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the native state (N) and functions in two retention mode including HIC and weak cation-exchange (WCX). In this thesis, the 2D column was initially employed for carrying out both of the renaturation of the reduced/denatured RNase A by two modes, respectively. The feasibility and advantages of this new type dual-functional matrix in the protein separation and protein refolding also were discussed. In addition, the active components in cord blood serum were scanned prelimianrily with three kinds of multidimensional chromatography coupled with MALDI-TOF MS in this thesis.This thesis contains four chapters Chapter I:A Literature Review. Overview of the development, progress and application of protein folding liquid chromatography method, as well as multi-dimensional chromatography in proteomics applications.Chapterâ…¡:The refolding of reduced/denatured RNase A was investigated by the new type dual-functional matrix with IEC mode. The effects of urea concentration in mobile phase, GSH/GSSG ratio, the pH value, folw rate, and protein concentration on the refolding efficiency and mass recovery of the reduced/denatured RNase A were investigated under IEC mode in detail. The results indicated that the aggregation of denatured protein can be diminished by adding low concertration of urea in the mobile phase, the disulfide exchange of reduced/denatured RNase A can be accelerated with the presence of GSH/GSSG at the ratio of 8:1 in the mobile phase. A better result can be obtained at higher flow rate of the mobile phase. Under the optimal refolding condition, the bioactivity and mass recoveries of denatured RNase A can reach to be 75.0% and 85.5%. A better mass recovery was obtained under IEC mode, and IEC can tolerate high concentrations of denatured-proteins.Chapter III:he refolding of reduced/denatured RNase A also was studied by the new type dual-functional matrix with HIC mode.The effects of urea concentration in mobile phase, GSH/GSSG ratio, the pH value and protein concentration on the refolding efficiency and mass recovery of the reduced/denatured lysozyme were investigated under HIC mode in detail. The results indicated that the aggregation of denatured protein can not only be diminished by 2.0 mol/L urea in the mobile phase, but the refolding efficiency and mass recovery of reduced/denturated RNase A can be increased. With the presence of GSH/GSSG at the ratio of 8:1 in the mobile phase employed at pH 8.0, the disulfide exchange of reduced/denatured RNase A can be accelerated resulting in forming the correct four disulfide bonds. Under the optimal refolding condition, the reducec/denatured RNase A can be renatured completely with HIC, and the bioactivity and mass recoveries of denatured RNase A with 8.0 mol/1 urea can reach to be 98.0% and 61.9%, while that of 7.0 mol/L GuaHCl-denatured RNase A to be 100.1% and 66.8%, respectively. A better bioactivity recovery were obtained under HIC mode.Chapterâ…£:The active components in cord blood serum were scanned prelimianrily with multidimensional chromatography (MDLC) coupled with matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). Firstly, the cord blood serum was pre-separated by MDLC, such as SEC-RPLC, SEC-HIC-RPLC, and HPSEC-RPLC SEC. After post-test activity, the active fractions by SEC were further purified by RPLC. Finally the molecular weights of the active fractions were detected with MALDI-TOF MS. The result indicated that one active component in cord blood serum with the molecular weight of 7756Da was obtained.
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