Practical implementation of aqueous two‐phase processes for protein recovery from yeast

A two‐stage extraction process for the recovery of intracellular proteins from brewers' yeast was selected as a practical model system to study the implementation of polyethylene glycol (PEG)–phosphate aqueous two‐phase systems (ATPS). Disrupted all suspensions generated by homogenisation and bead milling were used to study the impact of cell debris upon the partition behaviour of the intracellular products (bulk protein, fumarase and pyruvate kinase). Regardless of their origin debris particles did not significantly influence the partition behaviour of the intracellular products in selected ATPS distant from the binodal and at volume ratios greater than one. Recycling of used PEG into the initial extraction stage did not significantly influence the protein partition behaviour in batch ATPS. In the polymer recycling studies in continuous ATPS using spray columns, the addition of fresh materials to make up the deficits of phase‐forming chemicals compensate any negative effect of the continuous recycling of the top PEG‐rich phase. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. © 2000 Society of Chemical Industry     

The optimization of aqueous two‐phase extraction of lysozyme from crude hen egg white using response surface methodology

BACKGROUND: Aqueous two‐phase extraction is a versatile method for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG 4000/potassium citrate aqueous two‐phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG 4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized ATPS was proved to be at pH 5.5 and 30 °C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U mg^?1, 21.11 and 103%, respectively. CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme. © 2012 Society of Chemical Industry     

Novel approach for neuronal stem cell differentiation using aqueous two‐phase systems in 3D cultures

Stem cell therapy has emerged as a promising alternative for replacing lost cells involved in neurodegenerative diseases. High efficiency of differentiation and full cell viability are actual challenges to achieve the translation of cell therapies to the clinic. To address this, the construction of aqueous two‐phase systems in three‐dimensional (ATPS‐3D) cultures has been proposed. This technique involves the combination of two polymers in which cells are confined in dextran droplets immersed over a substrate located in a poly(ethylene glycol) phase. The controlled placement of cells in a defined pattern promotes intercellular communication. This review aims to provide insight into the techniques used to enhance neural differentiation and current challenges to achieve the implementation of cell therapies. Cell density, colony size, interconnectivity and an appropriate substrate to modulate paracrine signaling are factors that determine neural differentiation efficiency during the construction of ATPS‐3D cultures. Hence, this contact‐free technique enables the design of neural niches to recapitulate in vivo environments more accurately. © 2020 Society of Chemical Industry (SCI)     

Aqueous two‐phase systems for the recovery of a recombinant viral coat protein from Escherichia coli

In this study the use of an aqueous two‐phase system (ATPS) following the direct chemical extraction of a recombinant viral coat protein, from the cytoplasm of Escherichia coli, is evaluated. The driving force is the need to establish an economically‐viable process for the manufacture of a vaccine against human papilloma infection. The partition behaviour of recombinant L1 protein, the major structural protein of the virus, and DNA was investigated in a polyethylene glycol (PEG)–phosphate system. An evaluation of system parameters including PEG molecular mass and the concentrations of PEG and phosphate was conducted, to estimate conditions under which the L1 protein and DNA partition to opposite phases. ATPS extraction comprising a volume ratio of 1.00, PEG 1000 (18.0%(w/w)) and phosphate (15.0%(w/w)) provided the conditions for accumulation of DNA into the bottom phase and concentration of L1 protein into the opposite phase (ie partition coefficient of DNA; ln K_DNA < 0.0 and partition coefficient of L1; ln K_L1 > 2.5). The findings reported here demonstrate the potential of ATPS to recover recombinant protein released from E coli by direct chemical extraction. © 2002 Society of Chemical Industry     

Kinetic model for simultaneous cell disruption and aqueous two‐phase extraction

A process of simultaneous cell disruption and aqueous two‐phase extraction demonstrated improved product yield and selectivity compared with the traditional process of cell disruption followed by extraction. Addition of aqueous two‐phase components did not decrease the efficiency of cell disruption. Moreover, ADH, LDH and G6PDH recovery was enhanced in this new process with recovery ratios of 97%, 93% and 95%, respectively. Cell disruption kinetics were established based on a new mechanism that was different from traditional first‐order kinetics, consisting of the release of intracellular proteins and enzymes, the denaturation and subsequent renaturation of these proteins that may be due to the protection of the aqueous two‐phase components during the cell disruption process. To account for this, the kinetics parameters were calculated, and the experimental data were regressed. The resulting kinetic model could provide a better fit for the experimental data with a correlation ratio of 99% (for total protein), and led to insights into the differences between the new process and the traditional one. Copyright © 2005 Society of Chemical Industry     

Practical application of aqueous two‐phase systems for the development of a prototype process for c‐phycocyanin recovery from Spirulina maxima

A novel process for the recovery of c‐phycocyanin from Spirulina maxima exploiting aqueous two‐phase systems (ATPS), ultrafiltration and precipitation 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 PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the c‐phycocyanin and contaminants concentrate to opposite phases. PEG1450–phosphate ATPS proved to be suitable for the recovery of c‐phycocyanin because the target protein concentrated in the top phase whilst the cell debris concentrated in the bottom phase. A two‐stage ATPS process with a phase volume ratio (V_r) equal to 0.3, PEG1450 7% (w/w), phosphate 20% (w/w) and system pH of 6.5 allowed c‐phycocyanin recovery with a purity of 2.4 (estimated as the relationship of the 620 nm to 280 nm absorbances). The use of ultrafiltration (with a 30 kDa membrane cut‐off) and precipitation (with ammonium sulfate) resulted in a recovery process that produced a protein purity of 3.8 ± 0.1 and an overall product yield of 29.5% (w/w). The results reported here demonstrated the practical implementation of ATPS for the design of a prototype recovery process as a first step for the commercial purification of c‐phycocyanin produced by Spirulina maxima. © 2001 Society of Chemical Industry     

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

BACKGROUND: The potential use of plants as production systems to establish bioprocesses has been established over the past decade. However, the lack of efficient initial concentration and separation procedures affect the generic acceptance of plants as economically viable systems. In this context the use of aqueous two‐phase systems (ATPS) can provide strategies to facilitate the adoption of plants as a base for bioprocesses. Among the crops, soybeans (Glycine max) represent an attractive alternative since potentially they can produce high levels of recombinant protein. In this paper the processing of fractionated soybean extracts using ATPS is evaluated as a first step to recover recombinant proteins expressed in plants, using β‐glucuronidase (GUS; E.C. 3.2.1.31) as a model protein. RESULTS: The evaluation of the effect of system parameters provided the conditions under which the contaminant proteins from fractionated soybean extracts and GUS concentrated in opposite phases. A PEG 600/phosphate system comprising 14.5% (w/w) polyethylene‐glycol (PEG), 17.5% (w/w) phosphate, a volume ratio (Vr) equal to 1.0, and a system pH of 7.0 resulted in the potential 83% recovery of GUS from the complex mixture and an increase in purity of 4.5‐fold after ATPS. CONCLUSIONS: The findings reported here demonstrate the potential of ATPS to process fractionated soybean extract as a first step to isolate and purify a recombinant protein expressed in soybeans. The proposed approach can simplify the way in which recombinant proteins expressed in plants can be recovered. Copyright © 2007 Society of Chemical Industry     

Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system

Developing a novel Ionic‐liquid (IL) based aqueous two‐phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1‐butyl‐3‐methylimidazolium acetate+ potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of α‐amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three‐variable central composite design was employed to understand the effect of phase forming components on extraction studies of α‐amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting‐out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. © 2015 American Institute of Chemical Engineers AIChE J, 62: 264–274, 2016     

Modeling of two‐phase polymerization of acrylamide in aqueous poly(ethylene glycol) solution

Two‐phase polymerization of acrylamide (AM) has been successfully carried out in aqueous poly(ethylene glycol) (PEG) solution with 2,2′‐azobis[2‐(2‐imidazolin‐2‐yl)propane] dihydrochloride (AIBI) as the initiator. A new heterogeneous kinetic model has been developed based on the partitioning of components between the two phases. It was found that polymerization proceeded in both the continuous and dispersed phases, even though the latter was the dominating polymerization locus. Besides the initiator, monomer concentration, and polymerization temperature, the PEG concentration also significantly influences the polymerization rate. With increasing concentration of PEG, gel effects in the aqueous PAM droplets were enhanced and more monomer preferred to polymerize inside the droplets, hence, the polymerization kinetics accelerated. The proposed model can successfully predict the composition of each phase and the polymerization kinetics during the aqueous two‐phase polymerization over a wide range of various reactions conditions. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

New aqueous two‐phase systems based on poly(ethylene oxide sulfide) (PEOS) and potassium phosphate for the potential recovery of proteins

A new aqueous two‐phase system (ATPS) based on a degradable polymer called poly(ethylene oxide sulfide) with a molecular weight of 33 000 g mol^?1 (identified as PEOS‐12) and potassium phosphate was exploited for the potential recovery of proteins. An initial characterisation of the ATPS was achieved by the construction of a phase diagram for the PEOS‐12/phosphate system. The protein partitioning behaviour of lysozyme and bovine serum albumin (BSA), selected as single model proteins, and B‐phycoerythrin (BPE) produced by Porphyridium cruentum in the new ATPS under increasing tie line length (TLL) conditions at constant phase volume ratio (V_r) and system pH was investigated. Both single proteins partitioned in the new ATPS, initially exhibiting bottom phase preference; however, lysozyme changed phase preference when TLL was increased. Fractionation of a complex model (production of BPE by P. cruentum) using PEOS‐12/phosphate ATPS was performed to evaluate the potential protein recovery from fermentation broth or cell homogenate. The proposed new ATPS proved to be suitable for the potential recovery of BPE from crude extract of P. cruentum. In general, a system comprising V_r = 1.0, 18% (w/w) PEOS‐12, 8% (w/w) phosphate and 30% (w/w) TLL at pH 7.0 provided conditions to concentrate BPE into the bottom phase (i.e. partitioning behaviour of BPE; lnK_BPE = ?1.8) with a protein recovery of 84%. The findings reported here demonstrate the potential application of the new ATPS for the recovery of proteins from complex biological suspensions. Copyright © 2006 Society of Chemical Industry