Distribution Of Protein In Salting-Out Extraction System

Salting-out extraction (SOE), is an effective separation method that target substrace and impurity could disturbuted in organic solvent and salt-water phases based on their solubility differences. SOE has been used in separation of protein, such as lipase, recombinant human serum albumin, immunoglobulin G, amylase, cellubose enzymes and so on. The characteristics of SOE include higher yield and impurity removal ratio, protection toward protein structure and biological activity, mild operational condition, easy scale-up, rapid process, low cost. So SOE is expected to be an attractive technique with bright prospect in industrial production application. So far the rule of SOE, the factors which might influence protein distribution are still not well documented. The rule of protein distribution was summed up by various proteins at different conditions in this paper. The separated and purified human plasma protein with SOE combined chromatography was also studied.Firstly, the factors which effect the extraction ability of split-phase in natural SOE, such as different kinds of salts and organic solvents, pH, NaCl added, were investigated. In ethanol SOE system, the range order for salt in phase diagram was:potassium citrate<ammonium sulfate<potassium carbonate<dipotassium phosphate<sodium carbonate; in dipotassium phosphate SOE system, the range order for solvent in phase diagram was:methanol<ethanol <2-propanol<1-propanol<tertiary butyl alcohol<2-butanol; the range of phase diagram became narrow when the pH decreased:adding NaCl could narrow the concentration range of salt in ethanol/dipotassium phosphate SOE and ethanol/potassium citrate SOE significantly.Secondly, the factors in SOE which effected the distribution of bovine serum albumin (BSA), for example, the kinds of salts and organic solvent,the length of tie-line, pH, NaCl added, were studied. At the same time, the effects of the hydrophobicity and molecular weight of protein on protein distribution in SOE and the factors which influence phase formed time were investigated.BSA was likely to distribute into the top phase from bottom phase when the ionic strength of salt increased, but to the bottom phase when the organic solvent polarity decreased; the SOE system with short tie-line of various kinds of SOE, and closer to the critical point gave higher yield; adding3%(w/w) NaCl into ethanol/dipotassium phosphate SOE could greatly improve the yield of protein; hydrophobicity played great roll in protein distribution, hydrophobic protein tended to distribute to bottom phase, and hydrophilic proteins were more sensitive to organic solvent polarity in two-phase distribution. Finally, pH7,17.5%(w/w) ethanol/20%(w/w) dipotassium phosphate SOE system combined hydrophobic chromatography(HIC) and strong anion exchange chromatography were chosed to separate and purify human plasma proteins. The yield of HSA and IgG reached97.7%and95.4%in SOE, the high purity of HSA greater than98.56%by HPLC was obtained by one step HIC. IgG and remaining HSA could be separated and obtained as single band of SDS-PAGE by Gel-Pro analyzer software, the purity of HSA was99.28%and the yield of HSA and IgG reached92%,88%,respectively.