The Separation And Purification Of Plasma Protein And Recombinant Human Insulin By Salting-out Extraction

IgG and albumin are the main functional proteins in the plasma, which are used in the clinic for anti-virus, treatment of burn and bleeding. Insulin is the only hormone in the body to decrease the concentration of blood sugar, which is clinically used to treat diabetes. At present, organic solvent precipitation, multiple centrifugation and column chromatography method are mainly used in the separation and purification of proteins. These methods exist many problems such as complicated operation, long period, low protein recovery rate and easy to inactivate. This paper carried out the separation and purification of IgG and albumin in the plasma by the hydrophilic dissolved organic salting out agent extraction coupling column chromatography and the separation and purification of recombinant human insulin by the hydrophilic dissolved organic salting out agent extraction.Firstly, on the base of previous results,17.5% ethanol/20% K2HPO4 and 14% ethanol/20% K2HPO4 were enlarged to 500 g to study the stability of the salting out extraction effects. It is found that 14% ethanol/20% K2HPO4 was stable. Under pH 7.0, albumin and IgG were distributed in top phase and the yield rate were 90% and 65%, respectively. However, IgG was easy to precipitate under the 17.5% ethanol/20% K2HPO4 system. The conditions of hydrophobic chromatography and ion exchange chromatography were optimized to separate and purify the top phase of 14% ethanol and 20% K2HPO4 system. Under the optimized conditions, pure IgG was obtained by electrophoresis, but the purity of albumin was not enough high.Secondly, the distribution of plasma protein in 2-Methyl-2-propanol/salt system was investigated. The results showed that in 15% 2-Methyl-2-propanol/14% K2HPO4 system with pH 7.0 and 7.5% NaCl, BSA was distributed in the lower phase that the recovery rate is 98%, and IgG was distributed in the middle phase (interfacial precipitation) that the recovery rate is 77%, so albumin and IgG could be separated. The extraction phase and precipitation of 2-Methyl-2-propanol/K2HPO4 system were separated and purified by the optimized conditions of hydrophobic chromatography and ion exchange chromatography. The purity of IgG obtained was 100% by the analysis of electrophoresis and the purity of the albumin was 87%. This method was expected to be a new way to separate and purify plasma protein.In the end, the distribution of the recombinant human insulin in salting-out extraction system was investigated. The factors including the kinds of salts and organic solvent, the length of tie-line and pH which affected the distribution of recombinant human insulin was optimized. The best recovery rate of recombinant human insulin in top phase which was extracted by 18% ethanol/22% K2HPO4 with pH 9.0 was about 98%.