Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity

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• 作者：Yisheng Xu ; Malek Mazzawi ; Kaimin Chen ; Lianhong Sun ; Paul L. Dubin
• 刊名：Biomacromolecules
• 出版年：2011
• 出版时间：May 9, 2011
• 年：2011
• 卷：12
• 期：5
• 页码：1512-1522
• 全文大小：1101K
• 年卷期：v.12,no.5(May 9, 2011)
• ISSN：1526-4602

文摘

The effect of polyelectrolyte binding affinity on selective coacervation of proteins with the cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), was investigated for bovine serum albumin/尾-lactoglobulin (BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivity turbidimetric titrations were used to define conditions of complex formation and coacervation (pH_c and pH, respectively) as a function of ionic strength. The resultant phase boundaries, essential for the choice of conditions for selective coacervation for the chosen protein pairs, are nonmonotonic with respect to ionic strength, for both pH_c and pH. These results are explained in the context of short-range attraction/long-range repulsion governing initial protein binding 鈥渙n the wrong side of pI鈥?and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higher isoelectric point, inferred from lower pH_c, is shown to result from the negative 鈥渃harge patch鈥?on BLG, absent for BSA, as visualized via computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry (ITC). The relative values of pH for the two proteins show complex salt dependence so that the choice of ionic strength determines the order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, of BLG from a 1:1 (w/w) mixture with BSA was shown by SEC to provide 90% purity of BLG with a 20-fold increase in concentration. Ultrafiltration was shown to remove effectively the polymer from the target protein. The relationship between protein charge anisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, was tested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pH_c by 0.2, as anticipated on the basis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pH that was sufficient to provide enrichment by a factor of 2 for BLG-A in the coacervate formed from 鈥渘ative BLG鈥?