Integrated Process Of Salt-out Extraction And Column Chromatography For Separation And Purification Of Plasma Proteins

Plasma protein consist of hundreds of proteins with numerous physiological functions, and albumin and immunoglobulins of the plasma protein occuppy a great share of the blood products market. However, the separation and purification of them are quite difficult because of the complexity of blood. Organic solvent precipitation, salting-out and column chromatography are widely used to separate such products in industry, however, the disadvantages of these methods, such as low operation temperature, multi-steps seperation and low recovery rate, can not be ignored. In this thesis, an integrated method of hydrophilic organic solvent salting-out extraction(HOS-SOE) and column chromatography was developed to provide a new method for the separation and purification of blood proteins.Firstly, Tthe HOS-SOE system composed of diffrent concentrations salts (dipotassium hydrogen phosphate, sodium carbonateand sodium citrate) and hydorhpilic organic solvent (ethanol) were selected to extract IgG, and the distribution of IgG in top and bottom phase was studied, wherein the systems composed of20%dipotassium hydrogen phosphate (w/w)-14%ethanol(w/w),19%sodium carbonate (w/w)-13%ethanol (w/w) and25%sodium citrate (w/w)-19%ethanol (w/w) achived recovery rates of protein as high as97.44%,93.12%,90.23%, respectively. ELISA anaylsis showed that IgG activities remained unchanged in dipotassium hydrogen phosphate-ethanol system, howere, more than70%activity was lost in sodium carbonate-ethanol system.Secondly,14%ethanol(w/w)-20%K2HPO4(w/w)(pH7.0) system was chosen to extract IgG and albumin from pig plasma directely, under such condition of separation, glucose removal rate achieved51.82%, and some impurities protein also can be removed significantly. Taking the characteristics of salt-rich in top phase into consideration, hydrophobic interation chromatography was selected as subsequent pufication method. after the chrogatography, albumin and IgG were obtained in a mix manner with high purity, and then high purities of albumin and IgG could be obtained after the anion exchange chromatography, which shown a single band for each protein by non-reducing SDS-PAGE analysis, and over99%purity by densitometer scanning.The results showed that HOS-SOE combined with column chromatography technlolgy is an effective method in separation and purification of proteins from plasma. Compared with the saltiing-out mnethod,ethanol precpitation and chromatography techniqucs,the advantages of new method are ambient operation,short time in phase forming,high purity of albumin and IgG, simplicity in scale-up,the new method is expecte to be a alterative method in industrial application for plasma protein purification.