Extraction of β-galactosidase fused protein a in aqueous two-phase systems

A method for the purification of proteins hybridized with β-galactosidase and produced in Escherichia coli is suggested. The method is based on the dominating properties of the β-galactosidase part of the molecule that are utilized for extraction in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system. The purification of the hybrid protein Staphylococcal protein A-Escherichia coli-β-galactosidase (SpA-βgal) produced in Escherichia coli is described. The partitioning of the cell debris and SpA-βgal depended on the distance to the critical point, i.e., the length of the tie line. A poly(ethylene glycol) top phase and an interface free from cell debris were obtained for a composition close to the binodial with a relatively short tie line. At this composition no Spa-βgal partitioned to the interface. When the length of the tie line was increased, more of the SpA-βgal was caught by the interface. The partitioning of SpA-βgal to the top phase was also affected by the salts present during the extraction. The utilization of SpA-βgal for affinity extraction has been investigated. Experiments with SpA-βgal and fluorescence-labeled human IgG(hIgG-F) in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system showed that the complex SpA-βgal-hlgG-F was partitioned to the interface, probably as a precipitate.     

Partition of rat erythrocytes in aqueous polymer twophase systems

The partition of rat erythrocytes between the top phase and interface of aqueous poly(ethylene glycol)-dextran two-phase systems containing 0.15 M NaCl and 0.01 M sodium phosphate depends on the association of the cells with microscopic globules of dextran that persist in the poly(ethylene glycol)-rich top phase after the horizontal interface between the two phases has formed.     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

Combined use of extraction and genetic engineering for protein purification: recovery of beta-galactosidase fused proteins.

Partitioning of beta-galactosidase in aqueous two-phase systems of poly(ethylene glycol) and potassium phosphate is reviewed. The affinity of Escherichia coli beta-galactosidase for the PEG-rich phase dominates also in beta-galactosidase fusion proteins and the concept of using beta-galactosidase as an affinity handle for extraction of other proteins, after fusion, is discussed. A hypothesis is presented, assuming that tryptophan residues at the surface of beta-galactosidase is responsible for its partitioning to the PEG rich phase, and the concept of poly-tryptophan handles fused to the target protein for extraction is introduced.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

Lysozyme extraction from hen egg white in an aqueous two-phase system composed of ethylene oxide–propylene oxide thermoseparating copolymer and potassium phosphate

The recovery and purification of lysozyme from hen egg white has been investigated in an aqueous two-phase systems composed of thermoseparating random copolymers of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate. In the primary extraction step lysozyme was satisfactorily partitioned to the top polymer-rich phase in a system composed of 40% (w/w) EO₅₀PO₅₀, 10% (w/w) potassium phosphate, and 0.85 M sodium chloride at pH 9.0, diluted 3-fold with crude egg white, where contaminating proteins were discarded in the bottom phosphate-rich phase. After the primary phase separation the upper EO₅₀PO₅₀ phase was removed and subjected to temperature-induced (65 °C) phase separation, which resulted in the partitioning of pure lysozyme to the top water phase. The separation system was found to be efficient in achieving the purification of lysozyme in a high yield of 85% and specific activity of 32,300 U/mg of protein, with a purification factor of 16.9 and a concentration of lysozyme in the water phase of 2.3 g/l in two extraction steps.     

Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

The influence of phase volume ratio on partition and purification of penicillin acylase from Escherichia coli on poly(ethylene glycol)–sodium citrate aqueous two-phase systems was studied. In PEG 1000 systems both partition coefficients of the enzyme and total protein increased with decreasing phase volume ratio. However, in PEG 3350 containing NaCl, penicillin acylase follows a reverse trend, while total protein behaves in the same way. Implications for protein purification designs are discussed.     

Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase

Recombinant Escherichia coli (pATBX 1.8) secreting extracellular xylanase was used as a model system to study the application of an aqueous two phase system for extractive cultivation. An increase in the polymer concentrations from 6 to 20% in the polyethylene glycol phosphate aqueous two phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of xylanase in the top phase. However, varying phosphate concentrations from 8 to 16% decreased both the phase volume ratio and the partition coefficient of xylanase. The polyethylene glycol (6%) and phosphate (12%) system was found to be optimum for extracellular cultivation of E. coli, where extracellular xylanase was selectively partitioned to the top phase giving a purification ratio of above 1.0. The process was extended to a semicontinuous operating mode at the optimal condition, wherein the top phase containing xylanase was recovered and the surviving cells were recycled together with the new top phase. The maximum recovery of xylanase was obtained after 12 h in the top phase with a twofold increase in the specific activity as compared to the one obtained in the reference fermentation. In the present work, we report for the first time the use of the two phase system for the extractive cultivation of recombinant E. coli (pATBX 1.8) with the purpose of obtaining a simple and inexpensive separation procedure and achieving the maximal extraction of xylanase to one phase.     

Liquid–liquid extraction of a recombinant protein, cytochrome b5, from an impure extract using aqueous two-phase systems

The recovery and purification of a recombinant protein, cytochrome b₅, from an impure extract of Escherichia coli disrupted cells was carried out in one step using a liquid–liquid extraction process of aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts. With this separation process it was possible in one single step to remove the cell debris, that precipitate at interface of the system, and to obtain relatively high recovery yields, nearly 67%, of the target protein in the salt-rich phase, with purification factors up to 6.     

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.     

Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems

The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and system pH was carried out to determine under which conditions the laccase concentrates predominantly to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w), phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 95%. The results reported here demonstrated the potential application of ATPS for the valorisation of residual material and the potential establishment of a downstream process to obtain value added products with commercial application.     

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.     

Continuous extraction of β-d-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer

High concentrations of Escherichia coli disintegrate move the binodial of a poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase system towards lower concentrations. It has also been shown that the yield and purification factor of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) in the PEG phase was gradually improved by moving the experimental system to a composition closer to the binodial. The mass transfer rates of cell debris, total protein, β-d-galactosidase and DNA have been studied and were found to be fast enough to reach equilibrium between the phases after 1.9 s of mixing in a static mixer with 24 mixing elements. A continuous extraction process for β-d-galactosidase from E. coli has been designed on the basis of these studies with a mean residence time of 6.3 min from the disintegrator inlet to the β-d-galactosidase containing PEG-phase outlet of the centrifuge. This PEG phase contained 83.5% of the total β-d-galactosidase with a purification factor of 13.6, and only 2.8% of the total protease activity of the disintegrate. All cell debris and almost all DNA were confined to the potassium phosphate phase.     

Cultivation of Lactococcus lactis in a polyelectrolyte-neutral polymer aqueous two-phase system

The possibility to cultivate Lactococcus lactis in aqueous polymer two-phase system has been investigated. The phase system was made up of poly(ethylene imine) and (hydroxyethyl) cellulose. Long lag phases were needed for the microorganism to adapt to the polymer rich media. Cells favoured the (hydroxyethyl)cellulose rich top phase or they accumulated at the interface, while lactic acid showed affinity for the poly(ethylene imine) rich phase.Abbreviations PEG poly(ethylene glycol) - PEI poly(ethylene imine) - HEC (hydroxyethyl)cellulose     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Separation of clavulanic acid from fermented broth of amino acids by an aqueous two-phase system and ion-exchange adsorption

The clavulanic acid is a substance which inhibits the β-lactamases used with penicillins for therapeutic treatment. After the fermentation, by-products of low molecular weight such as amino acids lysine, histidine, proline and tyrosine are present in the fermented broth. To remove these impurities the techniques of extraction by an aqueous two-phase system of 17% polyethylene glycol molecular weight 600 and 15% potassium phosphate were used for a partial purification. A subsequent ion-exchange adsorption was used for the recuperation of the clavulanic acid of the top phase and purification getting a concentration factor of 2 and purification of 100% in relation to the amino acids lysine, histidine, proline and tyrosine.     

Liquid-liquid partitioning of some enzymes, especially phosphofuctokinase, from Saccharomyces cerevisiae at subzero temperature

The effects of low temperature (−18°C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied. The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at −18, 0 and (in some cases) 20°C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are 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.     

Coupling of poly(ethylene glycol) to albumin under very mild conditions by activation with tresyl chloride: characterization of the conjugate by partitioning in aqueous two-phase systems

Poly(ethylene glycol) activated with tresyl chloride has been covalently linked to albumin as a result of a 2-h incubation in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.125 M sodium chloride (0.344 OSM). The coupling of poly(ethylene glycol) to albumin was demonstrated by the increase in the partition coefficient of the protein in poly(ethylene glycol)-dextran aqueous two-phase systems. A linear relationship between the log of the partition coefficient of the poly(ethylene glycol)-albumin conjugate and the degree of modification (measured as the amino groups consumed during the coupling step) has been demonstrated. Countercurrent distribution in the two-phase system showed that poly(ethylene glycol)-albumin was heterogeneous with respect to its partitioning behavior, indicating that the albumin was not uniformly modified with poly(ethylene glycol).