Recombinant Human Granulocyte - Macrophage Colony Stimulating Factor Fermentation Process And The Liquid Chromatography Refolding With Simultaneous Purification

Refolding of denatured proteins by liquid chromatography (LC) is a newly developping technique. The denatured protein can be renatured wih simultaneous purification with LC.It can improve the renaturation efficiency of the target proteins, so it has been paid much attention in recent years. In this dissertation, the fermentation conditions and enlargedcraftwork for rhGM-CSF expressed by E.coli, were studied. The highly expressed and highly output rhGM-CSF can be obtained under the optimal fermentation condition. In addition, the renaturation with simultaneous purification of rhGM-CSF was investigatedwith high performance hydrophobic interaction chromatography (HPHIC), high performance iron exchange chromatography (HPIEC) and size exclusion chromatography (SEC).The dissertation includes five sections:1. ReviewSignificance of protein refolding is introduced briefly, and a comprehensive review on recently developed techniques of protein refolding is provided. Comment recent development of the refolding and purification for rhGM-CSF. It contains 99 references.2. Research of shaking-flask and enlarged craftwork for rhGM-CSFThe effects of culture media, culture temperature, time of inducing, expression times on the growth of bacteria and inoculum volume were investigated. The results showed the optimum conditions: temperature 32℃ for 4-5hours when the growth of bacteria was in the initial logarithm growth phase. Under these culture conditions, highly expressed and highly output rhGM-CSF can be obtained. The enlarged craftwork was also investigated.3. Renaturation and simultaneous purification of rhGM-CSF by HPHICHPHIC was applied to the renaturation with simultaneous purification of rhGM-CSF expressed by E. coli successfully. Effects of several factors, including stationary phase, salts, pH value, urea concentration and ratios of GSH/GSSG in the mobile phase on the renaturation and simultaneous purification of rhGM-SCF with HPHIC were investigatedrespectively. The results show that GSH/GSSG used can improve to form the correct disulfide bonds as a result that the renaturation efficiency of rhGM-SCF can be increased. In addition, in order to enhance the mass recovery of rhGM-SCF with HPHIC, the low concentration urea can be used in the mobile phase to prevent denatured protein aggregation. Under the optimal condition, rhGM-CSF can be renatured with simultaneous purification with HPHIC in 30 min only by one step, its specific activity, mass recovery and purity can be obtained to be 1.58 X 107IU/mg, 56.8% and 95.7% repectively4. Renaturation and simultaneous purification of rhGM-CSF by HPIECRhGM-CSF was also successfully renatured and purified simultaneously by HPIEC.The effects of the different pH values, the ratios of concentrations of GSH/GSS and urea concentrations in the mobile phase on the renaturation and simultaneous purification of rhGM-SCF with HPIEC were investigated respectively. The results show the the three factors have the remarkable effects on the renaturation efficiency and mass recoveryof rhGM-SCF. GSH/GSSG can improve to form the correct disulfide bonds as a result that the renaturation efficiency of rhGM-SCF can be increased. In addition, in order toenhance the mass recovery of rhGM-SCF with HPIEC, the low concentration urea can be used in the mobile phase to prevent denatured protein aggregation. Under the optimal condition, rhGM-CSF can be was renatured with simultaneous purification with HPIEC30 min only by one step, its specific activity, mass recovery and purity can be obtained to be 1.66x107IU/mg, 58.8% and 96.2%, repectively.5. refolding and purification of rhGM-CSF by SECrhGM-CSF was successfully renatured with partial purification by SEC. The effects of the different pH values, the ratios of concentrations of GSH/GSS and urea concentrations in the mobile phase on the renaturation and simultaneous purification of rhGM-SCF with SEC were investigated respectively. With the optimal conditions, the obtained rhGM-CSF has a specific activity of 1.31 xlO7IU/mg, purity of 86.4%, mass recovery of 42.3%. Compared to the SEC at a constant urea concentration, SEC with a urea gradient was more efficient, in terms of specific bioactivity and mass recovery.