Investigation Of Dissolving The Inclusion Bodies By Alkali Solution For Protein Folding Liquid Chromatography

The thesis consists of five chapters. Firstly, the recombinant human granulocyte colony-stimulating factor(rhG-CSF) was solubilized by a dilute alkaline solution, and the solubilized conditions were optimized. Then, the solubilized rhG-CSF at alkaline solution was proceeded by protein folding liquid chromatography (PFLC), including ion-exchange chromatography (TEC), immobilized metal chelate affinity chromatography (IMAC), hydrophobic interaction chromatography (HIC), respectively. Compare to the obtained mass recovery of rhG-CSF by these different kinds of chromatography, the mass recovery of rhG-CSF was increased. Recombinant human stem cell factor (rhSCF) was also solubilized at alkaline solution and refolded by PFSAX.1. The optimization of the solubilization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in alkaline solutionThe conditions for solubilizing the inclusion bodies of rhG-CSF in a alkaline were investigated systematically. It includes the pH, urea concentrations, concentrations of sodium hydroxide , and solubilization time and so on. The 0.1 mol·L^-1 sodium hydroxide solution containing 2.0 mol·L^-1 urea as the best condition for the solubilizing rhG-CSF inclusion bodies. The solubilized rhG-CSF inclusion bodies at alkaline solution was compared to the urea solution , the solubilized rhG-CSF inclusion bodies by alkaline solution shows that the latter has higher purity and higher concentration. After the solubilized rhG-CSF was precipitated by acetone, its purity improved.2. Refolding of rhG-CSF by PFLCThe .purification with simultaneously refolding of rhG-CSF by the several HPLC and obtained results are as following: (1) Weak anion exchange chromatography (WAX), purity of the rhG-CSF was 89.9%, specific bioactivity was 6.3 x 107 IU-mg"1 and mass recovery was73.0%. (2) Size exclusion chromatography and a WAX, purity and mass recovery were 96.5% and 71.5%, respectively. The specific bioactivity was 8.4x107 IU-mg'1. (3) Strong anion exchange chromatography (SAX), purity, 97.8%, specific bioactivity , 6.2 x 107 IU-mg"1, mass recovery, 58.0%. (4) Immobilized metal chelate affinity chromatography (IMAC), purity , 95.6%, specific bioactivity, 8.6 x 107 IU-mg"1, mass recovery ,76.9%. (5)Hydrophobic interaction chromatography (HIC), purity, 98.2% specific bioactivity was 7.6 x 107 IU-mg"1, mass recovery , 45.5%. Compared to usual solubilization of inclusion bodies by 8.0 mol-L"1 urea, the mass recovery of rhG-CSF by all the above PFLC modes was improved significantly.3. Solubilization, puriiication and refolding of rhSCF by SAX chromatographyThe recombinant human stem cell factor (rhSCF) was solubilized by the same composition of alkaline solution as that for rhG-CSF. For a comparison, the same inclusion bodies of rhSCF were also solubihzed by 8.0 mol-L"1 urea solution and then the two different solubilized rhG-SCF were refolded by strong anion exchange chromatography (SAX), respectively. For the former, purity, 96.3%, specific bioactivity, 7.84 x io5 IU-mg'1, mass recovery, 43.0%. for the latter, purity, 95.1%, specific bioactivity, 7.62 x 105 IU-mg"1, mass recovery, 36.4%. The results from the two methods for solubilizing inclusion bodies can be comparable with each other.