Aqueous two-phase extraction and purification of animal proteins

Aqueous two-phase systems provide a rapid, easily scalable method for separation of soluble proteins from insoluble materials and other undesired proteins. The method can be operated in continuous mode. It is particularly useful for animal proteins, as it overcomes difficulties of other methods in removing bulk insoluble material, while at the same time providing purification with respect to total soluble protein. This article describes the development of methods for aqueous two-phase extraction and purification of animal proteins, at both laboratory and pilot scale. The strengths, weaknesses, and possible future prospects for the method are discussed.     

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 extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

Aqueous two-phase extraction for protein recovery from corn extracts

Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.     

Dioxygen affinity in heme proteins investigated by computer simulation

We present an investigation of the molecular basis of the modulation of oxygen affinity in heme proteins using computer simulation. QM-MM calculations are applied to explore distal and proximal effects on O(2) binding to the heme, while classical molecular dynamics simulations are employed to investigate ligand migration across the polypeptide to the active site. Trends in binding energies and in the kinetic constants are illustrated through a number of selected examples highlighting the virtues and the limitations of the applied methodologies. These examples cover a wide range of O(2)-affinities, and include: the truncated-N and truncated-O hemoglobins from Mycobacterium tuberculosis, the mammalian muscular O(2) storage protein: myoglobin, the hemoglobin from the parasitic nematode Ascaris lumbricoides, the oxygen transporter in the root of leguminous plants: leghemoglobin, the Cerebratulus lacteus nerve tissue hemoglobin, and the Alcaligenes xyloxidans cytochrome c'.     

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.     

Aqueous two-phase extraction coupled with ultrafiltration for purification of amyloglucosidase

Aqueous two phase extraction (ATPE) in combination with ultrafiltration was employed for concentration and purification of amyloglucosidase produced by solid state fermentation. After extraction (with water) from dry moldy bran the dilute enzyme extract was concentrated by ATPE in a polyethylene glycol (PEG)/maltodextrin (MDX) system. The enzyme in the top PEG rich phase was then extracted into a Na₂HPO₄ rich bottom phase and further concentrated by ultrafiltration. The partitioning behavior of amyloglucosidase was examined in PEG/MDX, PEG/Na₂SO₄, PEG/Na₂HPO₄, PEG/KH₂PO₄ aqueous two phase systems. Effect of buffering salts such as NaCl, Na₂HPO₄, KH₂PO₄ and Na₂SO₄ on the partitioning behavior of enzyme was studied in PEG/MDX system. Maximum partitioning of amyloglucosidase was seen with KH₂PO₄ (m = 18.1). A two stage ATPE employing PEG/MDX (buffered with KH₂PO₄) and PEG/Na₂HPO₄ systems, followed by ultrafiltration has resulted in an overall recovery of 78.4% with 3.1 fold purification and 9.4 fold concentration of the enzyme.     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Purification of fusion proteins using affinity microspheres in aqueous two-phase systems

Affinity microspheres were prepared by immobilizing human -globulin (HGb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H; average diameter 0.33 m], which were prepared by emulsifier-free emulsion polymerization. HGB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced. Correspondence to: A. Kondo     

Purification of trypsin by affinity precipitation and combining with aqueous two-phase extraction

Summary Soybean trypsin inhibitor was bound to a pH-sensitive reversibly soluble-insoluble polymer (AS-L) for affinity precipitation of trypsin. Trypsin was purified from a crude extract 5.4-fold and with 61% yield, however, the reusability of the ligand was poor. This can be overcome by combining aqueous two-phase extraction with affinity precipitation, which improved trypsin yield and purity factor after repeated ligand usage.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

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.     

Aqueous two-phase assisted by ultrasound for the extraction of anthocyanins from Lycium ruthenicum Murr.

In this study, an efficient ultrasound-assisted aqueous two-phase extraction method was used for the extraction of anthocyanins from Lycium ruthenicum Murr. An ethanol/ammonium sulfate system was chosen for the aqueous two-phase system due to its fine partitioning and recycling behaviors. Single-factor experiments were conducted to determine the optimized composition of the system, and the response surface methodology was used for the further optimization of the ultrasound-assisted aqueous two-phase extraction. The optimal conditions were as follows: a salt concentration of 20%, an ethanol concentration of 25%, an extraction time of 33.7?min, an extraction temperature of 25°C, a liquid/solid ratio of 50:1 w/w, pH value of 3.98, and an ultrasound power of 600?W. Under the above conditions, the yields of anthocyanins reached 4.71?mg/g dry sample. For the further purification, D-101 resin was used, and the purity of anthocyanins reached 25.3%. In conclusion, ultrasound-assisted aqueous two-phase extraction was an efficient, ecofriendly, and economical method, and it may be a promising technique for extracting bioactive components from plants.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system

A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively, and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w) ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation processes.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system

Separation of 2,3-butanediol from the complex fermentation broths is a difficult task and becomes a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broths. Aqueous two-phase extraction of 2,3-butanediol from fermentation broths was studied by ethanol and dipotassium hydrogen phosphate system. The influences of phase composition on partition of 2,3-butanediol, removal of cells and biomacromolecules were investigated. The partition coefficient and recovery of 2,3-butanediol reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-butanediol to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. This process is convenient and economic, furthermore, the operation is easy to scale-up, that is, this method provides a new possibility for the separation and refining of 2,3-butanediol.     

Complexes of metal phthalocyanines with globin as the models of heme proteins.

The reaction between iron and cobalt tetrasulfonated phthalocyanines and globin results in the formation of the green complexes, as has been proved by difference spectroscopy. Spectrophotometric titration data indicate the formation of those complexes at the molar ratio 1:1. The complexes of ferrous, ferric and cobaltous tetrasulfonated phthalocyanines with globin have been isolated from the reaction mixtures by separation on Sephadex G-50 and precipitation of the protein fractions with ammonium sulfate. The visible spectra of these complexes are characterised by the main intensive peak at 641 nm, 678 nm, and 675 nm for ferric, ferrous and cobaltous derivatives, respectively. The new globin complexes have the property of reversible combination with oxygen and coordination with cyanide ions. It is evidence from the results of the spectrophotometric titrations of hemoglobin and methemoglobin with cobaltous tetrasulfonated phthalocyanine that iron protoporphyrins are displaced by this cobalt derivative; this suggests that phthalocyanine and porphyrin are bonded in a similar manner.     

PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems

Summary PEG has been activated using epoxy-oxirane, epichlorohydrin and periodate based reactions. The coupling to activated PEG of several protein ligands of different sizes was investigated. Glutathione, trypsin inhibitor, Protein A and anti-BSA have been bound to PEG and used to increase the selectivity of protein separation in aqueous two-phase systems.