Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells.

Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Partition of isomeric dipeptides in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems

A series of isomeric dipeptides, i.e., those containing identical residues but in different order such as Trp-Gly versus Gly-Trp, was partitioned in a poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase system. Dipeptides having a more hydrophobic character favored the upper (PEG) phase. Moreover, the partition coefficients for isomeric dipeptides are different, with the partition coefficients for dipeptides containing the more hydrophobic residue in the C-terminal position being, in general, greater than the partition coefficients for corresponding isomers which contain the more hydrophobic residue in the N-terminal position. These observations can be attributed to the different interactions that the isomers have with specific two-phase systems.     

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.     

Characterization of aqueous two-phase partition systems by distribution analysis of radiolabeled analytes (DARA): application to process definition and control in biorecovery.

In this article, we describe a characterization method applicable to aqueous two-phase systems (ATPS) heavily loaded with complex biological feed-stocks. We also studied the partition behavior of mixtures of traceable and quantifiable radiolabeled amino acids, selected on the basis of their relative hydrophobicity A unique linear relation was established between the tie-line length (TLL: commonly determined by graphical methods) and the hydrophobic factor (HF) for ATPS comprising potassium phosphate and PEG alone, and validated for polymer molecular weights from 300 to 8000 Da in systems operated at an apparent pH value of 7.5. Radiolabeled amino acids were subsequently applied to the characterization of ATPS loaded with whole bovine blood by the determination of effective tie-line lengths (TLL(e)). The addition of biomass to ATPS increased TLL(e) relative to that of blank ATPS of equivalent original composition of PEG and phosphate. In addition, an increase of biomass loading (variously sourced from blood, yeast, and E. coli) contributed to phase formation and stabilization of loaded ATPS in respect of system sensitivity toward operational conditions. The controlled application of sensitive ATPS (adjacent to the binodal curve) could thus be reconsidered for further application of aqueous two-phase partitioning as a primary purification process. The application of effective tie-line determinations by distribution analysis of radiolabeled analytes (DARA) as a process-aid in the design and operation of ATPS in biorecovery is discussed.     

An aqueous hydroxypropylstarch-poly(ethylene glycol) two-phase system for extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey.

The partitioning of alpha-lactalbumin and beta-lactoglobulin from bovine whey has been studied in an aqueous poly(ethylene glycol) (PEG)-hydroxypropylstarch two-phase system. The influence of several parameters including concentrations of polymers, sodium phosphate buffer, KSCN, and of PEG palmitate, with and without the presence of Ca2+, on the partitioning of the proteins has been investigated. The separation of the two proteins was demonstrated by counter-current distribution. A purification procedure for both proteins has been developed by using PEG-hydroxypropylstarch two-phase system. This system is compared with the more costly standard system based on PEG and dextran. The possible use of the aqueous two-phase systems for batch extraction for large scale purification of these whey proteins is discussed.     

Pancreatic serine protease extraction by affinity partition using a free triazine dye

Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit) - were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196% (w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.     

Column chromatographic separation of cells using aqueous polymeric two-phase systems

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multistep countercurrent distribution procedure has to be used. However, its operation is limited by time and apparatus constraints. As an alternative strategy we have developed a chromatographic technique in which the dextran-rich phase of a dextran/polyethylene glycol (PEG) phase system is immobilized onto derivatized agarose beads. The PEG-rich phase is used as the eluent. Inclusion of PEG-fatty acid affinity ligand gradients into the eluent produces separations of mammalian erythrocytes based on the differential interaction between the fatty acid and the erythrocyte membranes. A model separation of dog and human erythrocytes has been carried out.     

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.     

Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems

For various reasons extraction of proteins from plant material is difficult. In particular phenolic compounds and polyanionic cell-wall mucilages render conventional procedures of extraction and purification much more difficult. In this respect, aqueous polymer two-phase systems are presented as a powerful technique in extraction of vanadate-dependent bromoperoxidases from the brown macroalga Laminaria digitata, a seaweed extremely rich in mucilages. Little bromoperoxidase activity was obtained when fresh thallus material was extracted in Tris buffer. Extraction from freeze-dried and powdered material was more efficient but only satisfactory when partitioning in an aqueous polymer two-phase system was employed. Among several two-phase systems tested, one composed of poly(ethylene glycol) (PEG 1550) and potassium carbonate proved most successful (phase system-1). A rapid and efficient extraction procedure was developed with special regard for suitability in large scale processes. Staining for catalytic activity after PAGE revealed a pattern of several bromoperoxidase isoforms. Bromoperoxidases extracted in phase system-1 were fractionated into two groups of isoforms by partitioning in a second system (phase system-2) indicating that isoforms from both groups differ significantly in surface properties. Subsequently, one purification step by hydrophobic interaction chromatography was sufficient to remove residual non-peroxidase proteins as well as remaining polysaccharides from bromoperoxidases of both groups. Thus, consideration of aqueous two-phase systems as a technique for extraction and purification of plant proteins can be recommended, whenever inconveniant amounts of phenolic compounds, mucilages or pigments are present.     

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.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface hydrophobicity

Two different series of hydrophobically modified proteins were partitioned in a number of aqueous two-phase systems (ATPS) to investigate the effect of hydrophobicity as a single property on partitioning. The modified proteins were derived from beta-lactoglobulin and bovine serum albumin (BSA). Measurement of the surface hydrophobicity of the proteins is important; hydrophobic interaction chromatography (HIC) was used for this purpose. The resolution of the systems (R) in terms of protein surface hydrophobicity and the intrinsic hydrophobicity (log P(0)) of the systems was established. The effect of the addition of NaCl to PEG/phosphate and PEG/dextran systems was analyzed in terms of the hydrophobicity difference between the phases and their ability to promote hydrophobic interactions between the protein surface and the PEG molecules. The values for R and log P(0) differed somewhat depending on which group of modified proteins was used for partitioning. The addition of NaCl to PEG/phosphate systems promoted an increase in the values of R, showing an important effect on the resolution of the systems for protein surface hydrophobicity (twice as high when compared with systems without NaCl). For PEG/dextran systems, the addition of 9% NaCl (w/w) promoted an improvement in the resolution toward surface hydrophobicity with an increase of 60% on the value of R. (c) 1996 John Wiley & Sons, Inc.     

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).     

Characterization of aqueous two-phase systems based on polydisperse phase forming polymers: Enzymatic hydrolysis of starch in a PEG-starch aqueous two-phase system

A new method for determination of the binodial of an aqueous two-phase system, using spectrophotometric measurements of the turbidity, is described in this article. The method is especially designed to characterize phase systems composed of polydisperse phase components. It gives information about the area representing the transition from homogeneous solution to a two-phase system. The two-phase systems studied were first a conventional Dextran T40-polyethylene glycol 20M (PEG 20M) system, then a less-well-defined phase system based on PEG 20M and partially hydrolyzed starch. The PEG 20M-starch system could be changed with respect to the volume ratio between the phases with time using hydrolytic enzymes, and the possibility of using the glucose released from the starch polymer is pointed out. Then the system is transformed to an extractive biconversion where the bottom phase polymer also served as the substrate.     

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.     

Enzymatic hydrolysis of soluble starch in a polyethylene glycol-dextran aqueous two-phase system

Hydrolysis of soluble starch by glucoamylase and β-amylase was investigated as a model reaction in an aqueous two-phase system consisting of polyethylene glycol (PEG) and dextran (DEX). Changes in glucose concentration observed in the batch reaction experiments with glucoamylase were almost identical for the aqueous two-phase and pure water systems, showing that the enzymic reactions investigated were not influenced by the presence of PEG and DEX. The partition of β-amylase into the DEX phase was insufficient compared to that of glucoamylase. Hence, the former enzyme was crosslinked with glutaraldehyde to increase its apparent molecular weight and, as a consequence, the partition coefficient, defined as the concentration ratio of the component partitioned into the PEG phase to that into the DEX phase, was decreased to 17% of that of the original enzyme. In the operation in which the enzyme and substrate are partitioned selectively into the DEX phase and allowed to react there while the product, thus transferring to the PEG phase, is recovered, the aqueous two-phase system with a smaller partition coefficient provided longer operational stability.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Combined hydrophobic-metal binding fusion tags for applications in aqueous two-phase partitioning

In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG/salt system. The presence of both types of residues in the tag increased the partitioning greatly. Particularly, four engineered tags (H6, FH6, WH6, and YH6) containing a hexa-histidine sequence as well as different hydrophobic residues, all increased partitioning more than twice, reaching K values around 20, as compared to another construct (His6-GFP) containing an isolated hexa-histidine sequence. YH6, also proved be beneficial for protein expression.     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.