Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Protein refolding by reversed micelles utilizing solid-liquid extraction technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.     

Important parameters affecting efficiency of protein refolding by reversed micelles

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.     

Direct refolding of inclusion bodies using reversed micelles

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.     

Protein assay in reversed micelle solutions

Summary An adaptation of the Lowry (1951) method has been developed for quantifying proteins in organic reversed micellar solutions. Ethanol is added to improve the re-extraction of the proteins from the micelles and a centrifugation step is included to separate the organic phase.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Protein folding and protein refolding.

The functional three-dimensional structure of proteins is determined solely by their amino acid sequences. Protein folding occurs spontaneously beginning with the formation of local secondary structure concomitant with a compaction of the molecule. Secondary structure elements subsequently interact to form subdomains and domains stabilized by tertiary interactions. Disulfide bond formation, and cis-trans isomerization of X-Pro peptide bonds, as the rate-limiting folding reactions, are enzymatically catalyzed during protein folding in the cell. Although folding of domains is fast enough to occur cotranslationally in vivo, such vectorial folding on the ribosome is not essential for attainment of the native structure of a protein. Slow steps on the pathway to the functional protein structure are docking reactions of domains, association of subunits, or reshuffling reactions at the oligomer level. Aggregation as a competing side reaction is prevented, and the kinetic partition between competing polypeptide folding and translocation reactions is regulated by chaperone proteins binding to incompletely folded polypeptides.     

Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0.     

High pressure EPR studies of protein mobility in reversed micelles

We have investigated the effect of pressure on structural properties of subtilisin solubilized in reversed micelles of Tween-85/isopropanol in hexane. Electron paramagnetic resonance (EPR) spectra of spin-labeled enzyme indicate a reduction in spin-label mobility when the enzyme is transferred from aqueous solution to the microemulsion. One explanation for the spectral broadening is a change in the protein's active-site conformation and/or dynamics. However, over a W(0) range of 80 to 180, EPR spectroscopy could detect no change in the enzyme's environment, conformation, or molecular dynamics. The EPR spectra also contained a contribution from free spin label located in an environment with a polarity roughly between that of propanol and bulk water. No changes in the polarity surrounding the free spin label nor in the enzyme's structural properties were evident at pressures up to 10,000 psi. Previous work has demonstrated that pressure can be used to manipulate the size of some reversed micelles, and the EPR data indicated that for this system such pressure tuning of micellar properties will not adversely affect the structure of solubilized enzyme. (c) 1994 John Wiley & Sons, Inc.     

Design of surfactants suitable for protein extraction by reversed micelles

New surfactants have been synthesized for potential use in reversed micellar protein extraction operations. Preferential solubility of the surfactant in an aliphatic solvent such as hexane, heptane, or isooctane and the formation of reversed micelles accompanied with solubilization of significant quantities of water can be achieved by using strongly hydrophobic, twin alkyl chains as the hydrophobic moiety. Different surfactants having identical water-solubilizing capacities can have significantly different behavior in protein extractions, where extraction efficiency appears to be governed by the nature of the interfacial complex that forms between surfactants and proteins. Bulky surfactant chains provide a steric hindrance to the adsorption of the surfactant to the protein surface, thus inhibiting solvation of the protein/surfactant complex, and hence protein extraction. Under these conditions, a precipitate forms either in the bulk aqueous phase or at the interface. Surfactants that can form a close-packed complex with the protein are excellent protein-solubilizing agents. Dioleyl phosphoric acid (DOLPA) appears to be the best surfactant currently available for protein extraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 26-32, 1997.     

Tyrosinase activity in reversed micelles

The hydroxylase and oxidase activities of mushroom tyrosinase were studied in both sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane and cetyltrimethylammonium bromide (CTAB)/hexane/chloroform reversed micelles. The enzyme presented its highest activity when the water to surfactant molar ratio (W ₀) was 20 for both systems. When entrapped in the AOT reversed micelles, the enzyme activity decreased with the increase in AOT concentration at a constant W ₀, and the enzyme not only presented a higher reaction rate related to its oxidase activity but also a shorter lag period related to its hydroxylase activity. The relation between water activity and W ₀ revealed that enzyme activity in reversed micelles was more related to the size of the micelles which was determined by W ₀ and less to the water activity. Tyrosinase in CTAB reversed micelles showed potential for the analysis of o-diphenols.     

Protein transfer from an aqueous phase into reversed micelles. The effect of protein size and charge distribution

Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum = isoelectric point +0.11 x 10(-3) Mr -0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di-(2-ethylhexyl)sulphosuccinate (AOT) reversed micelles a similar relationship was found. The percentage of protein transferred could be related to the symmetry of charge distribution over the protein. This symmetry was expressed as the % of random electric moments on a protein that is larger than the effective electric moment of the protein (% S) [Barlow, D. J. and Thornton, J. M. (1986) Biopolymers 25, 1717]. The larger the value of % S, the more homogeneously the charges are distributed and the lower the percentage transfer.     

Development of a new reversed micelle liquid emulsion membrane for protein extraction

A new type of liquid emulsion membrane containing reversed micelles for protein extraction is introduced. A three-step extraction mechanism is proposed including solubilization, transportation, and release of the protein. The surfactants Span80 and sodium di(2-ethylhexyl)sulfosuccinate (AOT) are used to stabilize the membrane phase and to build up the reversed micelles, respectively. alpha-Chymotrypsin was used as the model protein. The condition in the internal phase inhibits the solubilization process of the already extracted protein back into reversed micelles. Concerning the solubilization, we studied the influence of the AOT concentration in the membrane phase and the ionic strength in the external phase. The extraction rate increases with higher AOT concentration and decreases with higher ionic strength. Using NaCl in the external phase led to better extraction results than using KCl. Maximum extraction results of 98% into the membrane phase and 65% into the internal phase were obtained. This condition retained 60% of the enzyme's activity. The concentration of KCl in the internal phase does not affect the solubilization rate but the release into the internal phase. By this way the ionic strength in the internal phase is used as the driving force for the protein release. The solubilization process is much faster than the diffusion and the releasing process, as found by variation of the extraction time. The influence of the operating conditions on the membrane swelling is also discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 267-273, 1997.     

Cell-free translation in reversed micelles.

Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.     

Protein refolding mediated by reverse micelles of Cibacron Blue F-3GA modified nonionic surfactant

An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery.     

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles.

The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by 1H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored.