Polymer recycling in aqueous two-phase extractions using thermoseparating ethylene oxide–propylene oxide copolymers

This is a study on the recovery and recycling of copolymer in aqueous two-phase systems containing random copolymers of ethylene oxide (EO) and propylene oxide (PO). The random copolymers separate from water solution when heated above the lower critical solution temperature (LCST). The primary phase systems were composed of EOPO copolymer and hydroxypropyl or hydroxyethyl starch. After phase separation the upper EOPO phase was removed and subjected to temperature induced phase separation. Copolymers with different EO/PO compositions have been investigated, EO50PO50 [50% EO and 50% PO (w/w)], EO30PO70 and EO20PO80. The temperature required for thermoseparation decreases when the PO content of the copolymer is increased. The effect on the recovery of copolymer after addition of salts, a second polymer or protein was investigated. The added components increased the recovery of copolymer after thermoseparation, e.g., increased the amount copolymer separated from the water phase after thermoseparation. Recycling of copolymer and measurements of polymer concentrations in the primary top and bottom phases after repeated recycling steps was performed. The fluctuation in polymer concentration of the phases was very small after recycling up to four times. Partitioning of the proteins BSA and lysozyme was studied in primary phase systems after recycling of copolymer. The partition coefficients of total protein and lysozyme was not significantly changed during recycling of copolymer. More than 90% of the copolymer could be recovered in the thermoseparation step by optimising the temperature and time for thermoseparation. In repeated phase partitionings in EOPO–starch systems the EO50PO50 copolymer could be recovered to 77% including losses in primary system and thermoseparation, which is equivalent to a total copolymer reuse of 4.3 times.     

Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning.

Synthesis of conjugates of the ethylene oxide/propylene oxide copolymer UCON 50-HB-5100 and the triazine dyes Cibacron Blue F3G-A and Procion Yellow HE-3G is described. The UCON-dye conjugate of Procion Yellow HE-3G is used as a ligand for affinity partitioning of glucose-6-phosphate dehydrogenase from bakers' yeast. The enzyme is first partitioned in a two-phase system composed of UCON, UCON-ligand and dextran, and the two phases isolated in separate containers. A small amount of salt is then added to the upper phase, which contains the UCON-ligand-enzyme complex, and the temperature increased above the cloud point of the UCON polymer to give a new two-phase system. The new two-phase system consists of an upper salt/water phase containing free enzyme and a lower UCON/water phase containing free UCON-ligand. Temperature-induced phase partitioning is thus seen to be of much assistance in dissociating enzyme-ligand complex, recovering enzyme and recycling UCON-ligand.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na₂SO₄.     

Hydroxypropyl cellulose/poly(ethylene glycol)-co-poly(propylene glycol) aqueous two-phase systems: system characterization and partition of cells and proteins.

Novel aqueous polymeric two-phase systems are described. These systems are formed by mixing hydroxypropyl cellulose (molecular mass 100,000, trade name Klucel L) with poly(ethylene glycol)-co-poly(propylene glycol) copolymer [molecular mass 6,500, poly(propylene glycol) content 50% w/w, trade name Pluronic P105], in a saline buffer. The phase diagram was measured and the interfacial tensions, phase separation times, and lower phase viscosities of three phase systems having constant Pluronic P105 concentration but varying in Klucel L concentration were determined. The partition behavior of a representative cell, bacterium, and protein and the affinity ligand-mediated alteration in the partition behavior of a protein from a yeast extract protein mixture were also characterized. The results suggest that Klucel L/Pluronic P105 phase systems may be cost-effective substitutes for, or complements to, existing aqueous polymeric phase systems. The physical characterization and representative partition data reported here should facilitate application of these new systems.     

Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C(14)H(29)) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12 degrees C in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.     

Separation of amino acids and peptides by temperature induced phase partitioning. Theoretical model for partitioning and experimental data

There is a strong interest in use of ‘smart polymers’ in separation systems. These are polymers which can react on external influence, such as temperature or pH change. With such polymers it is possible from the outside to affect the properties of a separation system. Amphiphilic copolymers show drastic changes in solubility properties, such as self-association and phase separation, at e.g. temperature increase. The random copolymers of ethylene oxide and propylene oxide units (EOPO-polymers) can form aqueous two-phase systems above the copolymer cloud point temperature. Two phases are formed, one consisting of 40–60% polymer in water and the other of almost 100% water. Amino acids and peptides can be partitioned in the thermoseparating systems. The partitioning strongly depends on the solute hydrophobicity, where aromatic amino acids and peptides are partitioned to the polymer phase and hydrophilic to the water phase. Salt effects can be used to enhance the partitioning of charged molecules. The thermodynamic driving forces which govern the partitioning of molecules in a thermoseparated aqueous phase system is described with use of the Flory-Huggins theory for polymer solutions. Expressions are derived which show the entropic and enthalpic effects on solute partitioning. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aqueous polymer two-phase systems formed by new thermoseparating polymers

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C₁₄) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.     

Effect of salts and surfactants on the partitioning of Fusarium solani pisi cutinase in aqueous two-phase systems of thermoseparating ethylene oxide/propylene oxide random copolymer and hydroxypropyl starch

An aqueous two-phase system composed by a thermoseparating random copolymer of ethylene oxide/propylene oxide 50/50 (%w/w), Breox, and hydroxypropyl starch – Reppal PES 100 was evaluated for the partitioning of Fusarium solani pisi recombinant cutinase. The effect of several additives on the partitioning of pure cutinase was evaluated. Micelles of sodium dodecanoate provided a ten-fold increase of the partitioning coefficient (K=9) and recovery yields of 60-75%. The phase diagrams of the systems composed of Breox, Reppal and sodium dodecanoate were determined and it was found that in systems with high surfactant concentrations, the binodal was moved to lower polymer concentrations, enabling a two-phase system with 6% (w/w) of each polymer.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems: recycling of thermoseparating polymer and surfactant with temperature-induced phase separation.

An effective system has been developed for purification of apolipoprotein A-1 from Escherichia coli fermentation solution and human plasma using aqueous two-phase extraction and thermal-phase separation. The system included non-ionic surfactants (Triton or Tween) and as top phase-forming polymer a random copolymer of ethylene oxide (50%) and propylene oxide (50%), Breox PAG 50A 1000, was used. The bottom phase-forming polymer was either hydroxypropyl starch, Reppal PES 100 and PES 200, or hydroxyethyl starch, Solfarex A 85. The top-phase-forming polymer and the surfactants are thermoseparating in water solution, i.e., when heated a water phase and a polymer/surfactant phase are formed. Recombinant apolipoprotein A-1, the Milano variant, was extracted from E. coli fermentation solution in a primary Breox-starch phase system followed by thermal separation of the Breox phase where the target protein was recovered in the water phase. Both in the Breox-starch system and in the water-Breox system Triton X-100 was partitioned to the Breox phase. The addition of non-ionic surfactants to the Breox-starch system had strong effect on the purification and yield of the amphiphilic apolipoprotein A-1. In a system containing 17% Breox PAG 50A 1000, 12% Reppal PES 100 and addition of 1% Triton X-100 the purification factor was 7.2, and the yield 85% after thermal separation of the Breox phase. Recycling of copolymer and surfactant was possible after thermal separation of copolymer phase. Approximately 85% of the copolymer and surfactant could be recycled in each extraction cycle. DNA could be strongly partitioned to the starch phase in the primary-phase system. This resulted in a 1000-fold reduction of E. coli DNA in the apolipoprotein A-1 solution obtained after thermoseparation. In extraction from human plasma containing low concentrations of apolipoprotein A-1, it was possible to reach a purification factor of 420 with 98% yield. By reducing the volume ratio to 0.1 Apo A-1 could be concentrated in a small volume of top phase (concentration factor 10) with a yield of 85% and a purification factor of 110.     

Enzyme purification using temperature-induced phase formation.

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.     

New polymers forming aqueous two-phase polymer systems

A new type of polymer, starch-modified by acrylamide, has been developed for application in aqueous two-phase systems (ATPS) for protein separation. Partial hydrolysis and acrylamide modification of starch to different degrees make it suitable for forming ATPS with poly(ethylene glycol) in a moderate concentration range. The potential of the polymer to form ATPS with the thermoprecipitating copolymer of 1-vinylimidazole with N-vinylcaprolactam (poly-VI/VCL) has been evaluated. The thermoprecipitation properties of poly-VI/VCL and Cu(II)-loaded poly-VI/VCL have been studied for application in metal affinity partitioning. The formation of ATPS with Cu(II)-loaded thermoprecipitating copolymer was critically achieved for poly-VI/VCL (10/90) copolymer in under-loaded metal concentrations. With the Cu(II)-loaded copolymer, poly-VI/VCL in the top phase and modified starch in the bottom phase, the ATPS formed was used for the purification of alpha-amylase inhibitor from wheat meal. The protein partitioned in the top phase and phase-separated polymer-protein complex could be precipitated by salt. The protein inhibitor was recovered with a yield of 75%.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I_core–hydrophobin I (EGI_core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI_core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI_core–HFBI was quantitatively back-extracted (K_{EGIcore–HFBI}=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI_core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI_core–HFBI into a water phase.     

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.     

Enhanced production of lactic acid through the use of a novel aqueous two-phase system as an extractive fermentation system

 In order to enhance the productivity of lactic acid and reduce the end-product inhibition of fermentation, the partitioning and growth of four different strains of lactic acid bacteria in three different aqueous two-phase systems were studied. Polyethyleneglycol/ dextran, polyethyleneglycol/hydroxypropyl starch polymer (HPS), and a random copolymer of ethylene oxide and propylene oxide (EO-PO)/HPS were used as polymer systems. One strain each of Lactococcus lactis subsp. lactis and of Lactobacillus delbrueckii subsp. delbrueckii partitioned completely to the interface and bottom phase in two-phase systems with low polymer concentrations of EO-PO/HPS100 and EO-PO/ HPS200. The growth and production of lactic acid by two of three L. lactis strains in a two-phase system with 5.5% (w/w) EO-PO and 12.0% (w/w) HPS100 were reduced by less than 10% compared with a reference fermentation in a normal growth medium. The viability of L. lactis subsp. lactis ATCC 19435 was maintained for at least 50 h and with four top-phase replacements during extractive fermentation in the EO-PO/HPS100 system. Moreover, when cell density reached the stationary phase in the first extractive fermentation, the lactate production in this aqueous two-phase system was maintained. Received: 2 October 1995/Received revision: 16 January 1996/Accepted: 22 January 1996     

Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.     

Hydrophobic surface properties of erythrocytes studied by affinity partition in aqueous two-phase systems

Summary Erythrocytes from various species have been partitioned in aqueous two-phase systems consisting of water, dextran, poly-(ethylene glycol), salt and buffer. The terminal hydroxyl groups of the latter polymer were esterified with palmitic, oleic, linoleic and linolenic acids, as well as with deoxycholic acid. In a two-phase system containing unesterified poly(ethylene glycol) the erythrocytes are exclusively in the dextran-rich lower phase. When the poly(ethylene glycol) is esterified the red blood cells collect at the interface and/or in the poly(ethylene glycol)-rich upper phase depending on the type and concentration of esterified acid. Palmitate ester is most effective in increasing the affinity of the cells for the upper phase, followed by oleate, linolate, linolenate, and deoxycholate esters. The partition behaviour of erythrocytes from various species differs considerably. Two groups can be distinguished: one consisting of erythrocytes from dog, guinea pig and rat, the other from human, sheep and rabbit. This division can be correlated to the content of sphingomyelin and phosphatidyl choline in the erythrocyte membranes.