On protein partitioning in two-phase aqueous polymer systems.

The partitioning of proteins between the coexisting phases of two-phase aqueous polymer systems reflects an intricate and delicate balance of interactions between proteins, polymers, salts and water. Experimental investigations have suggested that a large number of factors influence protein partitioning, including the types of polymers, their molecular weight and concentration; the protein sizes, conformation and composition; salt type and concentration, and solution pH; and the presence of ligands attached to the polymer which may interact with surface sites of the protein. Complementary modelling attempts have been successful in illuminating several molecular-level mechanisms influencing protein partitioning using lattice-model techniques, viral expansions and a scaling-thermodynamic approach. In spite of these experimental and modelling approaches, many of the physical phenomena associated with these complex systems are not well understood. Notably, the precise nature of the protein-polymer interactions and the potent effect of inorganic salts on the partitioning of proteins in these systems remains poorly understood.     

The molecular basis of partitioning in aqueous two-phase systems.

Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.     

Protein partitioning in two-phase aqueous polymer systems

Theories of protein partitioning in two-phase polymer systems which account for the effects of different aspects of system composition-such as the choice of materials, protein size, polymer molecular weight, polymer concentration, salt concentration, and affinity ligands-are reviewed. Although the present models provide some information about specific aspects of partitioning, a comprehensive and fundamental theory which can be used to predict protein partitioning behavior has not yet been developed. Some recommendations for future work are given.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Fundamental studies of biomolecule partitioning in aqueous two-phase systems

Phase diagram data at 4 degrees C was determined for the aqueous two-phase systems composed of polyethylene glycol, dextran, and water. The Flory-Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two-phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH(2) group on the c-terminal amino acid residue and by utilizing a set of low-molecular-weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH(2) group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low-molecular-weight proteins gave a linear relationship with the tie line compositions of a phase diagram.     

Purification of acid-proteases by affinity partitioning in aqueous two-phase systems

Summary An affinity polymer derivative was synthesized with the group specific acid protease inhibitor pepstatin attached to dextran (M.W. 500,0001). This derivative was used in an aqueous two-phase system with hydroxypropyldextran to purify crude solutions of chymosin and Endothia parasitica (EP) acid proteases. Chymosin was purified by a factor of 6.2 with an overall yield of 83%. EP protease was similarly purified. A new pepstatin binding protease was discovered in crude EP extracts.     

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand

The effect of the important ATPS- and bufferparameters on IgG and hybridoma partitioning in ATPSscontaining a PEG-dye-ligand was studied. Objective wasto establish selection criteria for effective ligandsfor extractive fermentations with animal cells inATPSs.In the presence of 1% PEG-dye-ligand the binding ofIgG to the PEG-ligand was affected severely by theNa-chloride concentration. The tie-line length and pHaffected IgG partitioning to a lesser extent. Thedesired partitioning of IgG into the top phase, wasonly obtained when, in addition to the 10 mmol/kgK-phosphate buffer, no Na-chloride was present. In anATPS culture medium, with ± 35 mmol/kg Na-bicarbonateand 60 mmol/kg Na-chloride, increasing thePEG-dye-ligand concentration up to 100% did increasethe partition coefficient, but was not effective inconcentrating the IgG in the top phase of ATPS culturemedium at a pH of 7.8.Furthermore, addition of the PEG-dye-ligand to ATPSculture medium changed the hybridoma cell partitioningfrom the bottom phase to the interface.     

Intensification of mass transfer in aqueous two-phase systems

A novel technique which intensifies conventional aqueous two-phase extraction by conversion of dispersed phase into colloidal gas aphrons (CGAs) has been developed for extraction of an enzyme. In the present work, amyloglucosidase (1,4-alpha-D-glucan glucohydrolase) was extracted using a polyethylene glycol-sodium sulfate-water system. The lighter phase, i.e., polyethylene glycol (PEG) rich phase, was converted into CGAs which were then dispersed into a salt rich phase. The effect of type of surfactant and its concentration, dispersed phase velocity, phase composition, and type of sparger on the dispersed phase mass transfer coefficient was investigated. The results suggests 9-16 times higher values of mass transfer coefficient compared to spray column. The multiorifice sparger at concentrations of 0.33 g/L of cetyl trimethyl ammonium chloride yielded best results. (c) 1993 John Wiley & Sons, Inc.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning

We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to beta-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient K(p) for these proteins was related to measured interfacial potential differences Deltaphi using the simple thermodynamic model, In K(p) = In K(o) + (F/RT)Z(p) deltaphi. The protein net charge Z(p) was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Z(p) deltaphi was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The beta-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The beta-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. (c) 1994 John Wiley & Sons, Inc.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Relationship between the protein surface hydrophobicity and its partitioning behaviour in aqueous two-phase systems of polyethyleneglycol-dextran

In order to develop possible correlations to predict partioning behaviour of proteins, five mammalian albumins (goat, bovine, equine, human and pig ones) with similar physico-chemical properties (molecular mass and isoelectrical point) were chosen. Evaluation of the relationship between hydrophobicity and partitioning coefficient (Kr) in polyethylenglycol-dextran (PEG-DxT500) systems formed by polyethyleneglycols of different molecular mass (3350, 6000 and 10,000) was investigated by estimating relative surface hydrophobicity (So) with a fluorescent probe, 1 anilino-8-naphthalene sulfonate. No relationship between Kr and So was found for systems formed by PEG3350, while aqueous two-phase systems with PEG6000 and PEG10,000 gave better correlations. The results obtained may be explained on the basis of an increase in the interaction between the latter PEGs and the protein due to their higher hydrophobic character which increases as the PEG molecular mass does so. In this way, systems with PEGs of higher molecular mass give the highest resolution to exploit hydrophobicity in partitioning.     

Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems

Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning.     

Influence of high concentration monovalent cations on the protein partitioning in polyethyleneglycol 1500-phosphate aqueous two-phase systems

The influence of chloride salts of Na+, Rb+ and Cs+ at concentrations from 0.15 to 1.2M was studied with bovine albumin, trypsin, ovoalbumin and lysozyme partitioning in an aqueous two-phase system formed by polyethyleneglycol 1500 and potassium phosphate at pH 7.4. Monovalent cations favoured the protein transfer to the polyethyleneglycol rich phase in the following order: Rb+ > Na+ > Cs+. Structure making cations as Na+ induced a poor loss of structured water, producing little diminution of the molar partial specific volume of polyethyleneglycol, while Rb+ and Cs+, structure breaking cations, induced a significant decrease in the specific volume of the polyethylene glycol. The increase of available solution free volume in the top phase favours the protein transfer to the polyethyleneglycol rich phase. Na+ and Rb+ induced a slight decrease in the alpha helix content of the proteins, while Cs+ increased the secondary structure for all the proteins. All the cations induced a decrease in the hydrophobic surface of the proteins, this effect was more significant in the presence of Cs+.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Charge directed partitioning

This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two-phase extration. The genetic modifications consisted of the fusion of charged peptide tails to beta-galactosidase and charge-change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl-dextran (DEAE-dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the beta-galactosidase, which carries a net negative charge, partitioned to the DEAE-dextran-rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein-rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient (K(p)) of up to 37-fold. However, these changes could not be solely attributed to charge-based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate-containing phase, and displayed four- to sevenfold shifts in K(p) as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of beta-galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2- to 15-fold shifts in K(p). (c) 1995 John Wiley & Sons, Inc.     

Statistical thermodynamics of phase separation and ion partitioning in aqueous two-phase systems.

A general model for the phase behavior of polymer-polymer aqueous two-phase systems containing small amounts of added inorganic salts has been developed from statistical thermodynamics. The model is based on the solution theory of Hill and new electrolyte solution model based on Fluctuation Solution Theory. It includes the effect of polymer molecular weight with scaling expressions from the Renormalization Group theory of polymer solutions. The model has been used to calculate the phase diagram and the partitioning of salt for an aqueous two-phase system containing polyethylene glycol (MW = 8000) and dextran (MW = 28,700) with 0.1 mole/kg of added Na2SO4. The calculations have been compared to experimental results with good agreement.