Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment.     

Aqueous two-phase protein partitioning using textile dyes as affinity ligands.

A simple and inexpensive aqueous two-phase system for the affinity partitioning of proteins is introduced. An aqueous solution consisting of maltodextrin (M100; molecular mass, 1800) and polyvinylpyrrolidone (PVP360; molecular mass, 360,000) formed two phases at 4 degrees C when the concentration of the polymers was 22.5% (w/w) and 4.0% (w/w), respectively. When the amino derivatives of chlorotriazine textile dyes or other azo textile dyes were added to the two-phase system they partitioned asymmetrically, favoring the upper, less dense, PVP360-rich phase. The association of the textile dyes with PVP360 did not prevent them from acting as affinity ligands for proteins. Three of the dyes screened increased the partition coefficient of purified lysozyme nearly 50-fold over a control containing no dye. Parameters such as pH, ionic strength, and dye concentration modulated the affinity-partitioning effect of the system. The partition coefficient of lysozyme in an egg white protein mixture increased severalfold as the total protein content of the system approached 4% (w/w), indicating that protein concentration is also important in determining the partitioning characteristics of this two-phase system. Proteins were efficiently freed of PVP360 and textile dye by recovery in a high-salt solution when another two-phase system was formed upon the addition of a solution of concentrated potassium phosphate to the isolated upper phase of a PVP360/M100/textile dye two-phase system. The affinity-partitioning system presented here allows one to screen large numbers of potentially useful protein ligands to optimize protein separation, followed by direct scaleup to a system size determined by the user.     

Protein separation using metal ion-bound particles in aqueous two-phase system

Metal ion affinity partitioning of protein in aqueous two-phase systems was studied using Sepharose as ligand carrier as an integrated adsorption partitioning. Cu(II)-bound Sepharose was mixed with protein solution and an aqueous two-phase system. The affinity sorbent was distributed quantitatively to the upper side or the interface. The binding studies of lysozyme to copper-bound gel in PEG/dextran two-phase systems demonstrate the feasibility of this bioseparation process. PEG/dextran system did not affect binding and elution of lysozyme to and from the Cu(II)-Sepharose particles.     

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.     

Metal affinity extraction of human hemoglobin in an aqueous polyethylene glycol-sodium sulfate two-phase system

Summary We have developed a protein extraction technique which uses metal affinity ligands in PEG/salt aqueous two-phase systems. Cu(II)IDA-PEG will partition proteins according to their surface histidine contents in two-phase systems formed from sodium sulfate and polyethylene glycol. The nearly complete separation of human hemoglobin and human serum albumin in a single stage is presented as a demonstration of the effectiveness of metal affinity extraction in PEG/salt systems.     

Partition of proteins in aqueous two-phase systems containing charged dextran or hydrophobic PEG

Summary The electrochemical effect of a charged dextran derivative and the hydrophobic effect of hydrophobic chain PEG derivative on partitioning of six types of proteins in PEG/dextran aqueous two-phase systems were investigated- When 1. 6%(w/w)DEAE-dextran was present in the system,the partition coefficient decreased quickly with increasing pH value;when 0. 4% (w/w)PEG pentadecanoic acid ester was present in the system, the partition coefficient of protein with strong hydrophobicity was greatly increased. The experimental results show that the influence of hydrocarbon chain PEG derivative on partition coefficient is closely related to the hydrophobicity of proteins.     

Vancomycin partitioning in aqueous two-phase systems: effects of pH, salts, and an affinity ligand

The partitioning of vancomycin in polyethylene glycol (PEG)-dextran and PEG-phosphate aqueous two-phase systems was studied at different pHs, at varying concentrations of neutral salts, and with an affinity ligand attached to methoxy polyethylene glycol (MPEG). Vancomycin is found to partition preferentially into the PEG-rich top phase, and its partition coefficient increases nearly exponentially with the addition of water structure-making salts, such as sodium sulfate and sodium chloride, but is independent of sodium phosphate concentration. In the PEG-dextran system the vancomycin partition coefficient increases 3-fold in acidic and neutral solutions, while in the PEG-phosphate system it increases about 30-fold on the addition of the same amount of sodium chloride (1. 5 mol/kg). In basic solution, above its isoelectric point, the vancomycin partition coefficient increases slightly with NaCI concentration in the PEG-dextran system. We also examined the use of the dipeptide D-ala-D-ala as an affinity ligand on MPEG to extract vancomycin into the PEG-rich phase. The vancomycin partition coefficient increased almost 7-fold upon adding the MPEG-ligand in an amount equal to approximately 3% of the total PEG in the system. Finally, fractionation of the polydisperse phase-forming polymers in the two-phase PEG-dextran system was observed. The effect of this polymer fractionation on the partition coefficient of vancomycin is discussed.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: I. Process concept and enzyme binding studies for pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase.     

Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system

Affinity extraction of dye- and metal ion-binding proteins, respectively, in a polyvinylpyrrolidone (PVP40)-Reppal PES 100 two-phase system was investigated. Due to the ability of PVP to complex azo dyes and inorganic ions, covalent coupling of the ligands was not essential. Cibacron Blue F3GA was used as the ligand for extraction of lactate dehydrogenase (LDH) from porcine muscle, while copper ions were used for extraction of B. stearothermophilus LDH with a fusion tag of six histidine residues (His6-LDH) from recombinant Escherichia coli homogenate. The binding strength of the enzymes to their respective ligands was only slightly reduced in the presence of PVP. The partition coefficient of Cibacron Blue and Cu2+ ions in the two-phase systems composed of different concentrations of PVP and Reppal was in the range of 20-30, with maximal partitioning being observed in the 17% (w/w) PVP40-10% Reppal PES100 system. Only a minor leakage of the ligands to the bottom phase was observed with time. The partitioning of porcine LDH to the PVP phase was increased 100-fold, and a maximal recovery of 89% was obtained in the two-phase system loaded with 0.2% (w/w) Cibacron Blue. The enzyme was quantitatively recovered with further purification from the PVP-dye phase using a secondary extraction step with 170 mM phosphate or alternatively with 100 mM phosphate containing NADH or NaCl. A more than 10-fold increase in the partition coefficient of His6-LDH was achieved in the two-phase system loaded with 0.4% (w/w) copper sulfate compared to the system lacking the metal ions. The enzyme was also back-extracted into phosphate phase in the presence of imidazole.     

Aqueous two-phase metal affinity extraction of heme proteins

An iminodiacetic acid derivative of poly(ethylene glycol) (PEG-IDA) that chelates metal cations has been synthesized and used to extract proteins in metal affinity aqueous two-phase PEG/dextran systems. With less than 1% of the PEG substituted with chelated copper, partition coefficients are shown to increase by factors of up to 37 over extraction with unsubstituted PEG. The proteins studied are preferentially extracted into the Cu(II)PEGIDA phase in proportion to the number of accessible histidine residues on their surface. The affinity contribution to partitioning is proportional to the number of exposed histidine over a very wide range. The partition coefficients of heme-containing proteins measured in the Cu(II)PEG-IDA/dextran systems increase with the pH of the extraction mixture from pH 5.5 to pH 8.0, while partition coefficients in the unsubstituted PEG/dextran systems are very nearly independent of pH. The strong pH dependence of the metalaffinity extraction can be utilized in the recovery of the extracted protein.     

Polymer fractionation in aqueous two-phase polymer systems.

We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.     

An improved purification procedure of alkaline phosphatase from calf intestine by applying partition in aqueous two-phase systems and dye-ligand chromatography.

Aqueous two-phase partitioning has been elaborated in order to improve the purification of alkaline phosphatase from calf intestine in larger scale. The laborious precipitation and centrifugation steps for the removal of the enzyme from the cell debris and from the bulk protein were replaced by this technique yielding a high recovery (88%) and a significant lower time requirement. For the preparation of 100.000 units (46 mg) of a homogeneous enzyme 2.0 kg of a system containing 200 g PEG 4000 and only 10 g dextran M 70 is necessary. Affinity partitioning in aqueous two-phase systems was used to screen 41 dyes for selecting a suitable ligand for the dye-ligand chromatography of the enzyme. In the case of alkaline phosphatase the results obtained by affinity partitioning coincide with the experimental requirements for the affinity chromatography of the enzyme. Procion Navy HE-R (Blue 171) exhibits a high affinity, selectivity and binding capacity for the enzyme compared with other dyes investigated. The purification procedure provided the same degree in purity (2200 U/mg) and yield (59%) if mucosa or chyme was applied as starting material. In the range of practical use the purified enzyme contains no detectable activities of DNAses (endonucleases) and DNA-nicking activities. The contamination with phosphodiesterase I (EC. 3.1.4.1.) is less than 0.01%.     

Dye-affinity techniques for bioprocessing: Recent developments

Textile or triazine dyes play an important role as affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in dye-affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-affinity chromatography has become a routine step in protein purification. New dyes have been developed and used successfully in both traditional chromatographic mode and new modes like affinity precipitation, polymer aqueous two-phase partitioning or expanded bed chromatography. The specificity of dye techniques has been increased by both purposeful designing of new dyes and decreasing non-specific protein–dye interactions with polymer shielding. One can envisage further development and ramification of dye-affinity techniuqes in protein purification.     

Partitioning of caseinomacropeptide in aqueous two-phase systems

This study evaluates the influence of type of salt and temperature on the partition coefficient of caseinomacropetide (CMP) to determine the best conditions for the recovery of CMP in aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG) 1500 and an inorganic salt (potassium phosphate, sodium citrate, lithium sulfate or sodium sulfate). In all systems, CMP presented affinity for the PEG-rich phase. The PEG1500+lithium sulfate showed the highest values of partitioning coefficient. In addition, thermodynamic parameters (DeltaH degrees , DeltaS degrees , DeltaG degrees) as a function of temperature, were calculated for the system PEG1500-sodium citrate at different PEG concentrations and the results imply thermodynamic differences between partitioning of CMP in this system.     

Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.     

Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides.

Polyethylene glycol (PEG)-coupled oligonucleotides are partitioned in an aqueous two-phase system PEG/dextran. The affinity of the oligonucleotide for the PEG-rich phase increases proportionally to the length of the coupled PEG polymer. After hybridization, the PEG-coupled oligonucleotide is able to force a complementary nucleic acid strand into the PEG-rich phase. This property can be used for the sequence-specific isolation of nucleic acids through hybridization-based affinity partitioning. The dependence of the partition coefficient in this system on various parameters is described. The application of this principle to multistage chromatographic separations is demonstrated.     

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.     

Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.