Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.     

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min⁻¹ at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

Partitioning studies of α-lactalbumin in environmental friendly poly (ethylene glycol)—citrate salt aqueous two phase systems

Aqueous two-phase systems (ATPS) formed by polymer and salt have been utilized to enrich the desired biomolecule into one of the phase with higher yield and purity. The eco-friendly, biodegradable poly ethylene glycol (PEG) and different citrate salts were chosen as ATPS phase components to investigate the partitioning behavior of α-lactalbumin (α-La). System factors and process parameters such as type and concentration of salt, molecular weight and concentration of PEG, pH, temperature and the effect of additives were studied and the results are discussed in detail. PEG 1000–tri-potassium citrate system yields high partition coefficient of 20 with a better yield of 98 % in the top phase. The addition of NaCl as an additive and acidic pH lowers the yield of α-La in the top phase. Influence of phase volume ratio (V _r) on partitioning was studied and found that the partition coefficient remains almost constant along the tie line. High yield was achieved at a V _r of 3.5 at the tie line length of 50.63 (%, w/w).     

ATPS applied to extraction of small molecules - polycetides - and simultaneous clarification of culture media with filamentous microorganisms

Aqueous two-phase systems (ATPS) were applied for extraction of small molecules (polycetides) - retamycin, an anthracyclin, and two red pigments, rubropunctamin and monascorubramin - from the whole culture media of Streptomyces olindensis and Monascus purpureus. ATPS allows, in one step, the separation of the small hydrophobic molecules in the PEG rich phase, from the filamentous microorganisms, which remains in the salt phase. Through experimental designs, the main variables and their levels were defined, as follows: for retamycin extraction, PEG 6000 (10%, w/w), phosphate at 20% (w/w) and pH 6.0 led to the higher partition coefficient, K(r) = 8.2, and yield = 91.3%; for red pigments, the statistical analysis indicate PEG 6000 (20%, w/w) and phosphate at 15% (w/w), for a high partition coefficient, (K(pig) = 113 and 150).     

Optimization of conditions for bacteriocin extraction in PEG/salt aqueous two-phase systems using statistical experimental designs

The bacteriocin nisin was extracted in PEG/salt aqueous two-phase systems (ATPS) using the property that the systems can extract hydrophobic proteins. The concentrations of the phase-forming components, PEG 4000 and Na(2)SO(4), were optimized for nisin recovery by means of statistical experimental designs, and it was found that they strongly influenced nisin recovery. The optimal composition of ATPS was found to be 15.99% (w/w) PEG 4000 and 15.85% (w/w) Na(2)SO(4) (pH 2), and the optimal ATPS allowed an 11.60% increase of nisin recovery compared to the standard method of nisin assay.     

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.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.     

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.     

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.     

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).     

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.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Isolation of polyhydroxyalkanoate from hydrolyzed cells of <Emphasis Type="Italic">Bacillus flexus</Emphasis> using aqueous two-phase system containing polyethylene glycol and phosphate

Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12%, w/v) and potassium phosphate (9.7%, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g%, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51% of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and ¹H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.     

Partitioning features of bovine trypsin and alpha-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

The partitioning of bovine trypsin and alpha-chymotrypsin--proteases of similar physico-chemical properties--in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.     

AQUEOUS TWO-PHASE EXTRACTION FOR THE PURIFICATION OF ALKALINE AGARASES FROM CULTURE EXTRACTS OF Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases—molecular weight of the PEG, system pH, system temperature, and NaCl concentration—were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Protein purification of Arthrospira platensis using aqueous two-phase system composed of polyethylene glycol and potassium phosphate/sodium citrate

Journal of Applied Phycology - Aqueous two-phase systems (ATPS) stand out as an alternative technique for recovering and concentrating proteins. However, the study of ATPS to recover Arthrospira...     

Oxidative renaturation of hen egg-white lysozyme in polyethylene glycol-salt aqueous two-phase systems.

Aqueous two-phase systems have been widely used for the separation and concentration of proteins. In this work we investigated the possibility of using aqueous two-phase system for the renaturation of inclusion body proteins by studying the effect of polyethylene glycol (PEG)-salt systems on the oxidative renaturation of hen egg-white lysozyme (HEWL) with guanidinium chloride (GdmCl) present in the system. To accomplish phase separation at moderately low concentrations of polymer and salt, the total GdmCl concentration had to be kept low (<1 M). The unfolded protein exhibited very low solubility under these conditions. In an attempt to increase the solubility of the protein, temperatures of 40, 50, and 60 degrees C were investigated. The effect of PEG molecular weight was also addressed. Best renaturation yields were obtained when using PEG 3400 and working at 50 degrees C. However, the total protein concentration had to be kept at a low level of 0.2 mg/mL. Lowering the total GdmCl concentration in the system resulted in increased aggregation.     

Effects of chemical modifications in the partition behavior of proteins in aqueous two‐phase systems: A case study with RNase A

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two‐phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono‐PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)–potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer‐rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG‐rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 378–385, 2013