Purification of phospholipase D by two-phase affinity extraction

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran-Ficoll, Dextran-PES, and Ficoll-PES two-phase systems, containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously and known solvatochromic solvent properties of the systems to characterize the protein-solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion-ion and ion-dipole interactions were characterized. These parameters, which are representative of solute-solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.     

Purification of papain by metal affinity partitioning in aqueous two‐phase polyethylene glycol/sodium sulfate systems

A simple and inexpensive aqueous two‐phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu²⁺ was attached to the polyethylene glycol‐iminodiacetic acid. The chelated Cu²⁺ content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase–forming components (polyethylene glycol 12–20% w/w and sodium sulfate 12–20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol‐iminodiacetic acid‐Cu²⁺ and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6‐fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.     

Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.     

Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system

Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics.     

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Extraction mechanism of sulfamethoxazole in water samples using aqueous two-phase systems of poly(propylene glycol) and salt

Based on the poly(propylene glycol)₄₀₀ (PPG₄₀₀)–salt aqueous two-phase system (ATPS), a green, economical and effective sample pretreatment technique coupled with high performance liquid chromatography was proposed for the separation and determination of sulfamethoxazole (SMX). The extraction yield of SMX in PPG₄₀₀–salt ATPS is influenced by various factors, including the salt species, the amount of salt, pH, and the temperature. Under the optimum conditions, most of SMX was partitioning into the polymer-rich phase with the average extraction efficiency of 99.2%, which may be attributed to the hydrophobic interaction and salting-out effect. This extraction technique has been successfully applied to the analysis of SMX in real water samples with the recoveries of 96.0–100.6%, the detection limits of 0.1 μg L⁻¹, and the linear ranges of 2.5–250.0 μg L⁻¹.     

Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems. Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-beta-Dglucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.     

Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.     

利用双水相分离回收离子液体的研究进展

研究离子液体的分离与回收对于减少离子液体对环境的影响、提高离子液体的利用效率、降低离子液体的应用成本、促进离子液体的工业应用具有重要的意义.本文重点综述了利用无机盐-离子液体双水相、糖-离子液体双水相、聚合物-离子液体双水相和CO2诱导的离子液体双水相技术分离回收离子液体的研究进展,分析了影响离子液体分离回收的关键因素,评价了不同离子液体双水相体系的优缺点,展望了该领域的发展方向及面临的挑战.     

Extraction and separation of a lysine-rich protein by formation of supramolecule between crown ether and protein in aqueous two-phase system

The macrocyclic calixarenes and crown ethers have recently been found to form hydrophobic complexes with the cationic protein cytochrome c (Cyt-c), by recognizing lysine residues on the protein surface. In the present study, it was found that the distribution of cytochrome c in Li₂SO₄/PEG aqueous two-phase system (ATPS) can be controlled by complexation with the crown ether dicyclohexano-18-crown-6 (DCH18C6). The protein was quantitatively extracted into the PEG-rich phase in the presence of DCH18C6 and perchlorate ion. Of various crown ethers and their analogues that were investigated, only DCH18C6 was able to extract cytochrome c into the PEG-rich phase. Extraction of cytochrome c in the ATPS using DCH18C6 is complete within 5 min. Cytochrome c complexed with DCH18C6 in the PEG-rich phase was quantitatively recovered into a salt-rich phase using K₂SO₄ by ion exchange of potassium ion and cationic protein in the cationic protein complex with DCH18C6. Selective extraction of cationic proteins was demonstrated in the ATPS. Under optimum conditions, the lysine-rich protein cytochrome c was selectively extracted over other cationic proteins using DCH18C6.     

Cd(II) extraction in PEG-based two-phase aqueous systems in the presence of iodide ions. Analysis of PEG-rich solid phases

Cd(II) plus iodide species were extracted into PEG-rich phases in the aqueous PEG(1550)-(NH₄)₂SO₄ system at pH 2.05–7.12. IR spectra show that increasing (NH₄)₂SO₄ solution acidity does not protonate PEG ether oxygen atoms, but decreases water content in the PEG-rich phases. Metallic species’ extraction into the PEG predominantly alters how water molecules bind to polymer chains; the changes in their absorption bands depend on pH. Microscopy shows that “fixation” of the extracted metal in the PEG-rich phase occurs by specific interactions which depend on the species. These also determine changes in the polymer chains’ conformation.     

Determination of trace alpha-fetoprotein variant by affinity adsorption solid substrate-room temperature phosphorimetry and its application to the forecast of human diseases

In the presence of ion perturber LiAc, 4-generation polyamidoamine dendrimers (4G-D) could emit strong and stable room temperature phosphorescence (RTP) signal at on nitrocellulose membrane (NCM), and Triton X-100 could sharply enhance the RTP signal of 4G-D. Triton X-100-4G-D was used to label concanavalin agglutinin (Con A) to get the labeling product Triton X-100-4G-D-Con A. Quantitative specific affinity adsorption (AA) reaction between Triton X-100-4G-D-Con A and α-fetoprotein variant (AFP-V) could be carried out on the surface of NCM, whose product Triton X-100-4G-D-Con A-AFP-V could emit strong and stable RTP and its ΔI_p was proportional to the content of AFP-V. According to the facts above, a new affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace AFP-V by Con A labeled with Triton X-100-4G-D was established. Detection limits of this method were 0.23 fg spot⁻¹ (direct method, corresponding concentration: 5.8 × 10⁻¹³ g mL⁻¹) and 0.13 fg spot⁻¹ (sandwich method, corresponding concentration: 3.2 × 10⁻¹³ g mL⁻¹). It has been successfully applied to determine the content of AFP-V in human serum and forecast human diseases, for its high sensitivity, long RTP lifetime, good repeatability, high accuracy and little background perturbation with at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V using AA-SS-RTP was also discussed.     

Liquid-liquid equilibrium of aqueous two-phase systems composed of poly(ethylene oxide) 1500 and different electrolytes ((NH4)2SO4, ZnSO4 and K2HPO4): Experimental and correlation

Phase diagrams of aqueous two-phase systems composed of PEO1500 + salt (di-potassium phosphate + potassium hydroxide or ammonium sulfate or zinc sulfate) + water were determined at (283.15, 298.15, and 313.15) K. All systems produce a large two-phase region; however the influence of temperature on the binodal position seems to be very small. By analyzing the effects of ammonium sulfate or zinc sulfate, it was observed that zinc was more effective in promoting phase separation than ammonium. The consistency of the tie-line data was ascertained by applying the Othmer-Tobias correlation. In this paper, aqueous two-phase systems data for nine ternary systems are correlated by using the NRTL model and UNIFAC for the activity coefficient. The results are very satisfactory, with root mean square deviations between experimental and calculated compositions as low as 0.99 and 1.21%, respectively. However the NRTL model better represents the systems in study, when compared with UNIFAC.     

Affinity partitioning of proteins in aqueous two-phase systems containing polymer-bound fatty acids. I. Effect of polyethylene glycol palmitate on the partition of human serum albumin and alpha-lactalbumin

The partition of serum albumin between the two aqueous phases of a polyethylene glycol-dextran-water two-phase system is strongly influenced by replacing a small fraction of the polyethylene glycol by its palmitate ester. The partition of albumin is compared with the partition of polymer-bound palmitate using polyethylene glycol [1-14C]palmitate. The data indicate that a maximum ("saturation") effect on the partition of albumin corresponds to a binding of fewer than two palmitate groups per albumin molecule. The effect on alpha-lactalbumin corresponds to the maximum binding of 0.5 palmitate group per protein molecule. These data do not fit the most favoured available model for affinity partitioning. The deviation can be attributed to the observation that the palmitate groups probably form micelle-type aggregates in the polyethylene glycol-rich phase at the concentrations used for albumin extraction.     

Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties.     

Phosphinoethyl-sulfonatoalkylthioethers and diphenyl-ω-sulfonatoalkyl-phosphines as ligands and polyoxyethylene–polyoxypropylene–polyoxyethylene triblock co-polymers as promoters in the rhodium-catalyzed hydroformylation of 1-dodecene in aqueous two-phase systems

The rhodium-catalyzed hydroformylation of 1-dodecene was investigated with a series of sulfonated water-soluble phosphine ligands at a pressure of 60 bar CO/H₂ and a temperature of 120 °C. Seven different groups of water-soluble phosphines were used for our investigations. We established an optimized ligand/rhodium ratio of 5 for the phosphines 1a, [Ph₂P(CH₂)₂S(CH₂)₂SO₃Na], and 1b, [Ph₂P(CH₂)₂S(CH₂)₃SO₃Na]. The utilized arylphosphino-thioether-alkylsulfonates formed with Rh(I) compounds highly active catalysts which could be recycled. The addition of detergents speeds up the hydroformylation reaction, but disturbs the phase separation (recycling). The best promotion effect and the smallest negative influence on phase separation gave polyoxyethylene–polyoxypropylene–polyoxyethylene triblock co-polymers. The ratio of 1-dodecene/rhodium could be increased up to 10.000 and we achieved turnover numbers (TONs)>50.000 without any surfactant and TONs of about 65.000 in presence of the co-polymers owing to the recycling on the catalytic system.