2011.03.17,Professors Sun Lianhong Published a paper entitled "Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity" in Biomacromolecules

  • Author:Yisheng Xu,MalekMazzawi,Kaimin Chen,Lianhong Sun,and Paul L. Dubin

    Abstract:The effect of polyelectrolyte binding affinity onselective coacervation of proteins with the cationic polyelectrolyte,poly(diallyldimethylammonium chloride) (PDADMAC),was investigated for bovine serum albumin/β-lactoglobulin(BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivityturbidimetric titrations were used to define conditions ofcomplex formation and coacervation (pHcand pHφ, respectively)as a function of ionic strength. The resultant phaseboundaries, essential for the choice of conditions for selectivecoacervation for the chosen protein pairs, are nonmonotonicwith respect to ionic strength, for both pHcand pHφ. Theseresults are explained in the context of short-range attraction/long-range repulsion governing initial protein binding “on thewrong side of pI” and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higherisoelectric point, inferred from lower pHc, is shown to result from the negative “charge patch” on BLG, absent for BSA, as visualizedvia computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry(ITC). The relative values of pHφfor the two proteins show complex salt dependence so that the choice of ionic strength determinesthe order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, ofBLG 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 chargeanisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, wastested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pHcby 0.2, as anticipated on thebasis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pHφthat was sufficient to provideenrichment by a factor of 2 for BLG-A in the coacervate formed from “native BLG”.

    dx.doi.org/10.1021/bm101465y | Biomacromolecules 2011, 12, 1512–1522

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