| HPLC is not only an important method for purification of biopolymers, but also a powerful tool for protein refolding. The chromatography packings are the core of HPLC, it plays a very important role for protein separation and protein folding liquid chromatography (PFLC). In this thesis, the refolding of reduced/denatured lysozyme was investigated initially by two dimensional chromatography packings with one single column, termed as 2D column, in weak cation exchange chromatography (WCX) and hydrophobic interaction chromatography (HIC) modes, respectively. In addition, the proteins from egg white wers purified with the 2D column offline, and the human pancreatic cancer biomarker from serum also was prepared with preparative-scale SCE column. This thesis includes the following four parts:1. Literature Review:This part mainly introduced the concepts and applications of two-dimensional liquid chromatography and single-column two-dimensional liquid chromatography, discussed the principle of protein refolding liquid chromatography, summed up the research progress of lysozyme refolding and early diagnosis of pancreatic cancer research, including a total of 64 references.2. The refolding of the reduced/denatured lysozyme was investigated initially by two dimensional chromatography with one single column, termed as 2D column, in weak cation exchange chromatography (WCX) and hydrophobic interaction chromatography (HIC) modes, respectively. The effects of urea concentration in mobile phase, GSH/GSSG ratio, the pH value, protein concentration and flow rate of the mobile phase on the refolding efficiency and mass recovery of the reduced/denatured lysozyme were investigated in detail with the two separation modes, respectively. The results indicated that using the 2D column, the reduced/denatured lysozyme can be renatured with the 2D column in WCX and HIC modes, separately. The bioactivity and mass recoveries of lysozyme were obtainded to be 94.6% and 96.0% in WCX mode at higher protein concentration (40 mg/mL), and 79.8% and 96.1% in HIC mode at lower protein concentration (5 mg/mL), respectively. As a result, The 2D column can be independently employed for accomplishing protein folding liquid chromatography (PFLC) in either WCX mode, or HIC mode. It was proved that this 2D column should be the first choice for accomplishing PFLC.3. Three proteins from egg white were separated and purified with 2D colomn offline, using one column initially. The egg white were separated in WCX mode firstly, and the lysozyme and advin can be purified with the purities of 95.3% and 95.6%, respectively. The fractions which were not retained in WCX mode on the 2D column were separated again in HIC mode with the 2D colume. Through conditional optimization,the protein G3 also can be purified successfully and the purity achieved to be 98.1%. However, ovomucin and conalbumin can not be separated complately. The results indicated that the two-demensional LC with a single colume can be used to separate biopolymers from the complex samples.4. Using 800.×40.0 mm I.D. preparative-scale size exclusion column, the pancreatic cacer serum sample was pre-separated, and then the fractions from SEC were further purified by RPLC. The biomarker of pancreatic tumor can be obtained with the molecular weight of 5734Da. Upon a further identification for the protein, it is expected to be a new tumor marker. Keywords:protein refolding; liquid chromatography; 2D column; reduced/denaturation; lysozyme; PanereatieCaneer; Biomarke...
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