Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems.

For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degrees C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.

Stimuli-responsive separation of proteins using immobilized liposome chromatography.

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s),max = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s),max = 11.3 at pH 4; carbonic anhydrase from bovine, k(s),max = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the ks-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.

Immobilized liposome chromatography for refolding and purification of protein.

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield.

Conformational transition and mass transfer in extraction of proteins by AOT--alcohol--isooctane reverse micellar systems.

We examined quantitatively the effect of alcohols on protein and reverse micellar structure. We used circular dichroism (CD) to compare the effects of various alcohols on the protein structure, and percolation phenomena to evaluate the effects of various alcohols on reverse micellar structure. Upon the addition of alcohols to the bulk aqueous phase, proteins were denatured significantly, depending on the alcohol species and concentration, suggesting that use of alcohol directly to the stripping solution is not effective in back-extraction processes of proteins. In the present study, a new method, a small amount of alcohol is added to the surfactant-organic solution to improve the back-extraction behaviors of proteins. Practically, in the back-extraction process, the alcohols suppressing the cluster formation of reverse micelles (high value of beta1), remarkably improved the back-extraction behavior of proteins. In addition, the same alcohol molecules showed a positive effect on the rate and fraction of protein back-extraction. From a result of the CD measurement of the back-extracted proteins, it was known that the alcohols added to reverse micellar solution allowed the proteins to back-extract safely without causing structural changes. These results show that the values of beta(t), defined by the variation of percolation processes, and the back-extraction behaviors of proteins have a good relationship, suggesting that the back-extraction processes were controlled by the micellar-micellar and protein-micellar interactions.

Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems.

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50-55 degrees C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60 degrees C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45 degrees C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

Oxidative refolding of lysozyme assisted by negatively charged liposomes: relationship with lysozyme-mediated fusion of liposomes.

Oxidative refolding of denatured/reduced lysozyme was examined in the presence of charged liposomes composed of neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Surface charge density of liposomes had a marked effect on the interaction between liposomes and reduced lysozyme which is observed in the early stage of the refolding. It was found that there was a critical level of surface charge density of liposomes (-0.15 C/nm2) at which the interaction between liposomes and lysozyme drastically changed. At the surface charge density of liposomes ranging from -0.15 to -1.4 C/nm2, the interaction between liposomes and lysozyme resulted in aggregate formation. In contrast, at the surface charge density ranging from 0 to -0.15 C/nm2, no aggregate formation was observed if the lysozyme/liposome molar ratio was less than 600. On the basis of the experimental results, a model for the interaction between charged liposomes and lysozyme was proposed, focusing on the mechanism of protein-induced fusion of charged liposomes as well as protein refolding on liposomes. Then, the optimal condition for oxidative refolding of lysozyme was examined in the presence of charged liposomes by controlling the lysozyme-liposome interaction. The reactivation yield of lysozyme was improved up to 85% in the presence of liposomes with a surface charge density of -0.14 C/nm2.

Dielectric response of cells and liposomes and its utilization for evaluation of cell membrane-protein interaction.

The dielectric measurements of an Escherichia coli (E. coli) cell suspension and liposome suspensions were carried out in the frequency range between 0.1 and 100 MHz to detect the heat stress-mediated interaction between proteins and cell membranes. The dielectric relaxation dispersion was observed to be above 1 MHz. The dielectric parameter (amplitude of dispersion, deltaepsilon) based on the Cole-Cole equation was anomalously changed with increasing temperature. The value of deltaepsilon of liposomes was varied at various temperatures depending on the type of protein present. The change in deltaepsilon of liposomes correlated with the amount of protein translocated across the phospholipid membrane. The changes in the value of deltaepsilon of E. coli cells with temperature variation was similar to those of liposomes in the presence of proteins, suggesting that the variation in dielectric parameters reflected the interaction between the phospholipid membrane and proteins. It was found that the dielectric measurement could be utilized for the detection of the interaction between proteins and liposomes.

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Oxidative refolding of Denatured/Reduced lysozyme utilizing the chaperone-like function of liposomes and immobilized liposome chromatography

Oxidative refolding of the denatured/reduced lysozyme was examined in the presence of small unilamellar vesicles (SUVs) essentially composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). SUVs facilitated the recovery of the enzymatic activity of lysozyme like molecular chaperones through the interaction with the refolding intermediate of lysozyme. The highest reactivation yield (87%) was obtained by delaying oxidation time (15-30 s) after dilution of the denaturant concentration was initiated in the presence of SUVs. SUVs supplemented with 1 mol % phosphatidylethanolamine were covalently coupled to gel beads, and then the interactions of SUVs with lysozyme at the various conformations were quantitatively examined with immobilized liposome chromatography (ILC). The reduced lysozyme lacking disulfide bonds was found to have a property similar to that of the molten globule state in terms of its local hydrophobicity, which was determined with the aqueous two-phase partitioning method. The reduced lysozyme was more clearly retarded on the ILC column than the native and 1 M guanidinium hydrochloride (GuHCl)-treated lysozymes with intact disulfide bonds. Similar results were obtained for alpha-lactalbumin, which has three-dimensional structure closely similar to that of lysozyme, regarding the membrane-protein interaction. These results can be interpreted as follows. In the early stage of the oxidative refolding, liposomes bound to the refolding intermediate of lysozyme lacking disulfide bonds. Then, liposomes assisted the formation of the tertiary structure of lysozyme by reducing protein intermolecular interactions, which usually cause the formation of the inactive aggregates. Consequently, the correct formation of the disulfide bonds was promoted. On the basis of the above results, the chromatographic oxidative refolding was also examined with ILC. The denatured/reduced lysozyme (9 mg/mL, 10 &mgr;L) was passed through the ILC column with a flow rate of 1 mL/min, which corresponds to the retention of lysozyme about 1 min in the ILC column. Subsequent oxidation of the eluted lysozyme resulted in the almost complete recovery (100%) of the original enzymatic activity of lysozyme.

Covalent immobilization of unilamellar liposomes in gel beads for chromatography.

For immobilized (proteo)liposome chromatography, unilamellar liposomes were covalently bound within gel beads that had been activated by CNBr, N-hydroxysuccinimide, tresyl, or chloroformate. Liposomes composed of phosphatidylcholine (PC) and 2 mol% of amino-containing lipid (phosphatidylethanolamine-caproylamine) were immobilized in the activated gels at 5-35 micromol lipid/ml gel and yields of 11-70%. The highest immobilized amount was found in chloroformate-activated TSK G6000PW gel, which contains large pore size (>100 nm). Liposomes composed of PC alone could also be attached to the chloroformate-activated gels at 33-42 micromol/ml gel and yields of 58-65%, probably by crosslinking of the phosphate moiety of phospholipid with the active group of the adsorbent. Liposomes prepared by various phospholipids with or without amino-containing lipids can generally be immobilized in the chloroformate-activated gels. The covalently bound liposomes were characterized by their high stability, unilamellarity, permeability of the membranes, and drug-membrane partition properties. A stable membrane phase was constructed for chromatographic experiments to be performed under extreme elution conditions.

Characterization and on-line monitoring of cell disruption and lysis using dielectric measurement.

The dielectric measurement of Escherichia coli suspension was carried out between 0.01 and 100 MHz during cell disruption using a physical method or cell lysis induced by chemicals for the purpose of on-line monitoring of cell disruption or lysis of bacterial cells. The dielectric dispersion of relative permittivity centered at about 1.4 MHz and the dielectric parameters based on the fitting calculation well reflected the damage of these cells under physical and chemical stresses. The degree of cell disruption as determined by the dielectric parameters, the amplitude of dispersion and the conductivity, well corresponded to those obtained by other conventional kinetic analyses. This methodology can be utilized for the on-line monitoring of physical cell disruption and cell lysis induced by detergent and could provide direct information to control these processes. Based on these results, the dielectric measurement was successfully applied to monitor the stress-mediated cultivation of E. coli cells.

Utilization of cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells.

The effective bioseparation process was developed by exploiting the cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells. At the observed optimal condition for heat stress (45 degrees C, 30 min) and that for chemical stress (20 mM Triton X-100, 5 mM EDTA, 30 min), E. coli cells were exposed to these stresses either simultaneously or sequentially. The operational sequence highly affected the yield and selectivity of the target recovery. Exposing the cells to the sequential stress (heating at 45 degrees C for more than 30 min after Triton X-100/EDTA treatment for 30 min) was found to be most effective for selective recovery of target beta-galactosidase among possible sequences.

Immobilized liposome chromatography for studies of protein-membrane interactions and refolding of denatured bovine carbonic anhydrase.

Small unilamellar vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1 mol% phosphatidylethanolamine were covalently coupled to chromatographic gel beads. Interactions of liposomal lipid bilayers with several water-soluble proteins, which had been denatured or partially denatured by 0.1-5 M guanidinium hydrochloride (GuHCl), were studied on gel beads containing the immobilized SUVs. The partially-denatured proteins treated with 0.5-1.0 M GuHCl were significantly retarded on the immobilized liposome column, whereas little retardation of native or unfolded proteins treated by >2 M GuHCl was observed on the same liposome columns. The retardation on the immobilized liposome column was found to be well correlated with local hydrophobicity, which was determined by the aqueous two-phase partitioning method using 1 mM Triton X-405 as a hydrophobic probe. It implies that the partially-denatured proteins are likely in a molten-globule state and associated with liposomal lipid bilayers. Chromatographic refolding of denatured bovine carbonic anhydrase (CAB) was achieved on the immobilized liposome column. The enzymatic activity of an unfolded CAB treated by 5 M GuHCl was recovered up to 83% after passing it through immobilized liposome column, whereas only 58% of the enzymatic activity was recovered when the denatured CAB was run on a liposome-free column. The refolding process is probably involved in the interaction of molten-globule state of CAB with the liposomal lipid bilayers.

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes.

Heating of several protein solutions at 40-47 degrees C for 5-60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of beta-gal at various temperatures were maximized under suitable heating conditions (45 degrees C, 30 min). Those of alpha-glu and CAB were maximized at 40-45 and 60 degrees C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of beta-gal, alpha-glu and CAB.

Enzyme release from heat-stressed cell membranes as a function of hydrophobicity evaluated by using aqueous two-phase systems.

The release behavior of a periplasmic enzyme, acid phosphatase, from heat-stressed Escherichia coli cells was characterized by using kinetic analyses when the cells were treated by Triton X-100-EDTA. The hydrophobicity of the cell surface and the release-rate of the enzyme were not influenced by heat treatment at temperatures between 30 and 50 degrees C. However, these values varied above 55 degrees C. The release-rate constants were found to correspond to the net and local hydrophobicity of the outer membrane surface, evaluated by aqueous two-phase partitioning.

Heat-induced translocation of cytoplasmic beta-galactosidase across inner membrane of Escherichia coli.

The behaviors of heat-induced translocation of cytoplasmic beta-galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules. The heat stress was found to induce translocation of cytoplasmic beta-galactosidase (beta-gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates. The translocation of beta-gal was then analyzed using (a) the location factor of beta-gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of beta-gal activity in periplasm after solublizing the outer membrane of E. coli cells by lysozyme/EDTA treatment. The LFG values were maximized when cells were stressed at the temperature of 42-47 degrees C. From the results on the surface properties of both beta-gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric beta-gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat-induced translocation of beta-gal across the inner membrane. The possibility to apply the heat-induced translocation of beta-gal for the enhancement of the target selectivity at the process upstream is finally presented.

Model system for heat-induced translocation of cytoplasmic beta-galactosidase across phospholipid bilayer membrane.

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.

Extractive cultivation of recombinant Escherichia coli using aqueous two-phase systems for production and separation of intracellular heat shock proteins.

The extractive cultivation of recombinant Escherichia coli cells to produce, release, and separate heat shock proteins (HSPs; GroEL and GroES) using poly(ethylene glycol) (PEG)/dextran (Dex) aqueous two-phase systems was developed. The growth rate of E. coli OW10/pND5 cells in the PEG/Dex two-phase media was almost the same value as that in the control media. The addition of 0.1 M potassium phosphate salts (KPi) increased the productivity of HSPs with keeping the growth rate of E. coli cells relatively high. The partition coefficients of HSPs were improved to greater values when phosphate salts were added at a concentration of more than 0.1 M. As a result, PEG/Dex systems supplemented with 0.1 M KPi were found to be the optimized two-phase systems for the extractive cultivation of E. coli cells. In the systems, the HSPs were selectively partitioned to the top phase while cells occupied the bottom phase and the interface between the two phases. This integrated process was extended to a semicontinuous operating mode, where the top phase containing the HSPs was recovered following intermittent heating and ultrasonic irradiation. The bottom phase containing cells and cell debris was recycled together with new top phase solution to repeat production and recovery of HSPs.

Extractive cultivation of Escherichia coli using poly(ethylene glycol)/phosphate aqueous two-phase systems to produce intracellular beta-galactosidase.

Escherichia coli cells were found to grow in poly(ethylene glycol) (PEG)/phosphate aqueous two-phase systems by selecting the phase-forming components and their concentrations, the tie-line length, and the phase volume ratio properly. The cells cultivated up to an optical density at 660 nm of 1.0 were disrupted by ultrasonic irradiation to release and recover overproduced beta-galactosidase. The surviving cells were found to grow immediately after ultrasonic irradiation. The PEG/phosphate (KH2-PO4-K2HPO4) system with added Na2SO4 was the one optimized for extractive cultivation of E. coli cells, where beta-galactosidase was selectively partitioned to the top phase while total soluble proteins and cells partitioned to the bottom phase. This integrated process was extended to a semicontinuous operating mode, where the top phase containing beta-galactosidase was removed following intermittent ultrasonic irradiation and the bottom phase containing cells was recycled together with the new top phase solution to repeat production and recovery of beta-galactosidase.

Increased activity of Chromobacterium viscosum lipase in aerosol OT reverse micelles in the presence of nonionic surfactants.

Modification of AOT reverse micellar systems with various alkyl glucosides and nonionic surfactants was attempted in order to improve the activity to Chromobacterium viscosum lipase. Tweens and Tritons, having the poly(oxyethylene) chain, have been shown to be solubilized at the oil-water interface of AOT micelles. Alkyl glucosides were also solubilized at the interface. These additives form mixed micelles with AOT and increase the concentration of the micelles. In contrast, Spans were found to be solubilized in the micro water pool of AOT micelles. Addition of nonionic surfactants decreased the hydrophobicity of the reverse micellar systems. Improvement of the lipase activity was achieved by the addition of nonionic surfactants, because of suppression of the hydrophobic and electrostatic interaction between AOT and lipase. The addition of nonionic surfactant having a poly(oxyethylene) chain has been shown to effectively increase the activity of the lipase in reverse micelles.