Polyelectrolyte-protein complexes precipitation

The ability of polyelectrolytes to remove oppositely charged proteins from solutions has been exploited through the incorporation of protein-polymer precipitation steps into a variety of protein purification procedures, wherein precipitated proteins are recovered from the insoluble complex aggregate via redissolution by pH or ionic strength adjustment (14-16). Furthermore, preferential complexation of polyelectrolytes with specific proteins has been substantiated (13). Although there are reports of the optimization of bulk complexation yield through the adjustment of solution parameters such as pH and ionic strength (17), very little has been accomplished in the optimization of the selectivity of complex formation. 

The ionic strength at which protein-protein complexes disintegrate is in general lower than for polyelectrolyte complexes. Probably because of the low charge density of the protein molecules and their less optimal packing within the complex. In some systems, an increase in temperature results in disintegration of the precipitates [82, 88]. This effect can be attributed to hydrophobic interactions and hydrogen bond formation between the amino acids. These interactions are known to be dependent on the temperature. 

In the study of BSA-PDMDAAC complex [34], it was found that the initial complexation pH is insensitive to the concentration of BSA and PDMDAAC. However, the coacervate of the complex formed at phase separation is not reversible at low BSA PDMDAAC ratio. For several protein-polyelectrolyte complexes, Kokufuta [9, 20] found that the amount of polyelectrolyte needed to precipitate a protein is linearly proportional to the amount of the protein in solution. The concentration dependence for the efficiency of protein-polyelec-trolyte phase separation was also reported by Morawetz [46] for several other systems. 

Upon the addition of polyelectrolyte to a protein solution, an increase in turbidity is observed due to the formation of an insoluble polyelectrolyte/pro-tein complex, followed by precipitation at a specific polyelectrolyte/protein mixing ratio. Such a change in turbidity is often accompanied by an increase in the measured solution conductivity (Fig. 18.3), which is particularly pronounced... 

The adsorption of DNA films assembled from oligonucleotides composed of two homopolymeric diblocks (polyA G and polyTnCn) were studied in the presence of salt. The growth of fihn increased with salt concentration [22]. The studies on polyelectrolyte complexation have offered wide applications such as water treatment, surface modification, dmg delivery system, tissue engineering. To understand the formation of protein-polyelectrolyte complex is important due to the interaction between polyanions or polycations with protein macromolecules or polyelectrolytes. Soluble complexes can be formed and amphorous can be precipitated with the interaction of molecules. Complex formation is generally performed in the bulk solutions. Potentiometry, conductometry, viscosimetry, turbidimetry, or electrophoretic and quasi-elastic light scattering are used to follow... 

Abstract Natural and synthetic polyelectrolytes have acquired notable importance in recent years due to their increasing application in different areas. One of these is downstream process methods which include the recovery, separation, concentration and purification of target enzymes from their natural sources. Polyelectrolytes interact with proteins to form soluble or non-soluble complexes. The interaction is driven by experimental variables of media such as pH, protein isoelectrical value, polyelectrolyte pKa, ionic strength and the presence of salts. The concentration of polyelectrolytes necessary to precipitate a protein completely is of the order of 10 " - 10 % p/v. Precipitation of protein by PE is a novel technique integrating clarification, concentration and initial purification in a single step. This chapter presents some properties of aqueous solutions of natural and synthetic PE as a tool to use them in the protein downstream process. 

Earlier work (9) has shown that the size of the precipitate, but not the protein recovery, depends on the method of addition of the polymer to the protein solution. Mixing conditions in the precipitation vessel also affect the precipitate size (lOh The solubility of the protein-polyelectrolyte complex depends strongly on the solution conditions—pH, ionic strength, polymer dosage level, and the nature of the protein and polyelectrolyte. These factors are discussed below ... 

Solution dH Several investigators (2, 12, 13) have indicated that the solution pH is an important determinant in the precipitation efficiency, and the optimum pH level will vary with both the protein (12) and the polyelectrolyte (5). However, the optimum pH for precipitation of protein by CMC did not change with the degree of substitution (DOS) of the CMC (12). This dependence is expected for the formation of an electrostatic complex. Changes in pH will affect the charge on the polyelectrolyte and the charge distribution on the protein. 

A model of protein-polyelectrolyte complexation as the result of electrostatic binding between charged protein and multiple binding sites of opposite charge on the polyelectrolyte has proven capable of describing most features reported here up to the point of optimum precipitation (18). 

The interaction between oppositely charged polyelectrolytes in aqueous environment leads to the formation of PECs. These PECs meet the profile of requirements of biocompatible polymer systems and can be adapted to meet the various requirements like carrier substances and components for active substances. The initial studies on macromolecular complexes were reported by Kossel. He described the formation of nucleoprotein complex due to interaction of proteins with nucleic acid and form heavy precipitates when mixed even at very high dilutions. In 1949, Fuoss and Sadek °... 

Apart from specific interactions between a target protein and a ligand-polymer conjugate, nonspecific interactions of protein impurities with the polymer backbone could take place. The nonspecific interactions limit the efficiency of the affinity precipitation technique, and significant efforts were made to reduce these interactions. The advantage of polyelectrolyte complexes as carriers for affinity precipitation is low nonspecific coprecipitation of proteins when the polymer undergoes a soluble-insoluble transition (10). 

Dissing U, Mattiasson B. Polyelectrolyte complexes as vehicles for affinity precipitation of proteins. J Biotechnol 1996 52 1-10. 

Polyanion-polycation-complexes are known for a long time on an empirical basis from the mutual precipitation of proteins. Already at the end of the previous century Kossel [1] recognized the electrostatic interaction between the oppositely charged polyions as the driving force for precipitation. Willstaetter [2] also introduced the term symplexes for polyelectrolyte complexes. 

For lysozyme precipitation, the protein content of the precipitate calculated from a mass balance is shown in Fig. 16.5. Protein concentration decreases with increasing PAA dosage, indicating the incorporation of more polyelectrolyte into the complex as polymer dosage is increased. 

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