Isolation And Purification Strategy Of Target Protein In Binary-and Ternary-protein Mixture On Column Chromatography

In this thesis the purification and isolation of target protein in complicated protein mixture was abstracted to the process of isolation and purification of binary-and ternary-protein mixture in order to investigate the strategy of isolation and purification on column chromatography.The feasibility of the purification strategies are verified through the instance of the purification of high purity hemoglobin from porcine whole blood and the isolation of low molecular weight hemoglobin from glutaraldehyde-polymerized porcine hemoglobin,in order to offer a high efficient and feasible program for the purification of target proteins at a large scale during the stage of pilot plant test.An instance of the purification of porcine hemoglobin and lysozyme on CM Sepharose Fast-Flow cation exchange chromatography have been carried out to study the purification strategy of binary mixture.In chromatogram typeâ… effective column capacity is primarily considered in flow through elution,so that a small amount of protein can be enriched.In chromatogram typeâ…¡adsorption mode of step gradient elution is primarily used,so as to shorten purification time.An instance of the purification of porcine hemoglobin,lysozyme and trypsin on SP Sepharose Fast-Flow strong cation exchange chromatography have been taken to study the purification strategy of ternary mixture.These strategies can be summed up as follows:one step of flow through elution for purification strategyâ… ;step gradient elution for purification strategyâ…¡;two steps of flow through elution for purification strategiesâ…¢. Using above strategies,the purity of purified porcine hemoglobin and lysozyme were analysed by gel exclusion high performance liquid chromatogram and ion exchange high performance liquid chromatogram were all above 99.9%and 95.0%,respectively.The recovery was all above 93.0%,and target product can be enriched in different extent.The output of new purification strategies is ten times more than traditional one,which shortened the purification time and increased the output per unit time.In the study of obtaining high purity hemoglobin purification from porcine whole blood, through optimizing buffer,pH,ionic strength,and dynamic column capacity and comparing the purification outcome of flow through elution and step gradient elution,chromatographic condition was finally choosed as follows:DEAE Sepharose Fast-Flow as stationary phase, 20.0 mmol/L PB+0.16 mol/L NaCl(pH8.0) as mobile phase to flow through elution untile impurity saturate the chromatographic column,then use 20.0 mmol/L PB+1.0 mol/L NaCl (pH8.0) to elute impurity.The flow rate is 76.4 cm/h.Purity of obtained Hb is 99.99%and 97.86%,which is analysed by TSK-GEL G3000SWXL and Shim-Pack WAX-1, respectively.Phosphatidylserine and phosphatidy lethanolamine have not been detected.All these have reached the United Stated FDA hemoglobin-based red blood cell substitutes quality standards of raw materials.In the study of isolation of low molecular weight hemoglobin from glutaraldehyde-polymerized porcine hemoglobin,three experiments was carried out and to compare the outcome of Sephadex G-75,hollow fiber column ultrafiltration and DEAE Sepharose Fast-Flow.Anion exchange chramotography was chose to optimize procedure.The chromatographic condition was as follows:buffer A was 20.0 mmol/L PB+0.06 mol/L NaCl(pH7.3),buffer B was 20.0 mmol/L PB+1.0 mol /L NaCl(pH7.3),firstly, chromatographic column was balanced by buffer A,then the sample was loaded,and the low molecular weight hemoglobin was eluted,finally,polymerized porcine hemoglobin was eluted by buffer B.The tetramer hemoglobin in purified polymerized porcine hemoglobin was lower than 5.0%,which was meet labortary quallity standard.These two examples fully proved the feasibility of the purification strategies.