Chinese Journal of Chemical Engineering, 16(6) 949 955 (2008) REVIEWS

New Development of Reverse Micelles and Applications in Protein Separation and Refolding*

LIU Yang ( ), DONG Xiaoyan ( ) and SUN Yan ( )**Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Abstract Reverse micelles bring mild and effective microenvironments in organic solvent that contain bio-molecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non-ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential appli-cation of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out. Keywords reverse micelles, ionic surfactant, nonionic surfactant, affinity, protein separation, protein refolding

1 INTRODUCTION

Reverse micelles are self-organized aggregates formed by surfactants in organic solvent, and nanometer- sized water pools are formed by the solubilization of water in their polar cores. The reverse micelles that are mostly with spherical shape are usually formed in ternary surfactant-water-organic solvent mixtures in-cluding surfactants ( 10%), water (0 10%), and or-ganic solvent (80% 90%), so the reverse micelles are also called water-in-oil emulsions, namely, Winsor II emulsions [1]. Reverse micelles are generally smaller than their hydrophilic counterparts (micelles), and their aggregation number being commonly lower than 50 [2]. Moreover, reverse micellar systems are colloi-dal solutions, so characteristic properties of these sys-tems are thermodynamic stability (no phase separation with time), spontaneous formation, low interfacial tension ( 10 2 mN·m 1), transparent nature (nano-meter size 100 nm), large surface area (102 103 m2·cm 3), viscosity comparable with pure organic solvents, and highly dynamic (constantly a collision and a fusion with each other, occasionally the fusion surfactant molecules and the contents inside reverse micelles exchange) [3].

The microwater pools inside reverse micelles are stabilized by surfactant monolayer within an organic continuum, which can solubilize hydrophilic bio-molecules such as proteins, enzymes, DNA, and amino acids. In reverse micellar systems, the biomolecules inside the polar core of surfactant monolayer are pro-tected from denaturation by organic solvent. Therefore, protein solubilization in reverse micelles plays a key role in a number of topics of biotechnology research. Nowadays, reverse micelles can be used as reaction systems for enzymatic catalysis [4], models of mem-brane systems separation of proteins [5], solvent-based extraction of proteins [6], microsurrounding for pro-tein structure discovery [7], and protein refolding [8, 9].

In the past three decades, the application of reverse micelles for bioseparation has attracted considerable attention because the technique is considered to be potentially useful in downstream processing for a large-scale separation of biomolecules from fermenta-tion mixtures.

Luisi et al.[10] first pointed out the possibility of the reverse micelles solubilization of proteins for pro-tein separation. Then, Dekker [11] and Goklen [12] developed this process systematically for the purpose of practical use. Later on, many researches demon-strated the factors such as water content (the molar ratio of water to surfactant, namely, W0) and micelle size [13], aqueous phase pH and ionic strength [14], surfactant type and concentration [15], and cosurfac-tant [16] effecting the protein solubilization based on the interactions between reverse micelles and proteins. Among these parameters, the surfactant is well known to play an important role in stabilizing protein solubi-lization in reverse micelles. It is also generally recog-nized that few current surfactants can form reverse micelles suitable for protein solubilization. Moreover, adding different proteins to the same reverse micelles can alter surfactant self-assembly and phase behavior [17]. Therefore, it is necessary to develop the new and well known reverse micellar systems for optimization of protein solubilization. In this article, the recent de-velopment of the various reverse micellar systems for protein solubilization that is composed of different surfactants including ionic surfactant-based reverse micelles, nonionic surfactant-based reverse micelles, mixed reverse micelles, and affinity-based reverse micelles is reviewed. Further, the characteristic prop-erties of the novel reverse micelles that consisted of the new surfactants are discussed. Moreover, the ap-plications of the reverse micellar systems in down-stream processing of biotechnology, especially the use in protein separation and refolding, are also summa-rized. Finally, it is pointed out that the universal affinity-

Received 2008-05-05, accepted 2008-09-17.

* Supported by the National Natural Science Foundation of China (20676098). ** To whom correspondence should be addressed. E-mail: ysun@tju.edu.cn

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based reverse micelles for protein separation and re-folding should be developed in downstream processing of biotechnology.

2 IONIC SURFACTANT-BASED REVERSE MICELLES

2.1 Conventional ionic surfactants for reverse mi-celles

Ionic surfactants as amphiphilic molecules are often used to form reverse micelles for protein solubi-lization, such as anionic di-2-ethylhexyl sodium sul-fosuccinate (AOT) [18] and cationic cetyl trimethyl ammonium bromide (CTAB) [19]. Saturated hydro-carbons are used as the organic solvents, such as hex-ane, isooctane, benzene, and cyclohexane. By choos-ing an appropriate set of experimental factors (pH, temperature, ionic strength, cosurfactants, and other parameters), it is possible to transfer a protein from a bulk aqueous phase to the water pool of reverse mi-celles in an organic phase (forward-extraction process) and later recover these proteins in a fresh aqueous phase (back-extraction process) [20]. By controlling these parameters, the extraction process can be varied via protein-micelles electrostatic, hydrophobic and steric hindrance interactions. Among these interactions, electrostatic interactions between the ionic surfactant molecules and the counter charge of the protein molecules are considered as the main driving force in forward extraction processes. Therefore, pH and ionic strength that mainly affect the charge numbers of pro-teins are dominant factors for the extraction process. Unfortunately, back extraction of proteins is not a simple reversible process of forward extraction in view of dynamics and thermodynamics [1]. There are two main problems in back extraction processes. The first one is the decrease of back extraction yields or activity yields in the processes, and the second one is that the rate of back extraction is much lower than the rate of forward extraction [21]. The former problem originates from the structural change of proteins and micelles due to the strong interaction between proteins and micelles, whereas the latter one is caused by the greater interfacial resistance to release a protein at the oil-water interface in the back extraction. In order to improve the back extraction process, many investiga-tions have been carried out using various methods. The strategy of improvement can be divided into three categories. One deals with the stripping aqueous phase by pH, species, and concentration of salts [22], the second deals with the surfactant-organic phase by spe-cies and concentration of surfactant or adding various alcohols [23], and the third deals with the whole sys-tem by temperature or pressure [20, 24]. In these methods, the first one is superior to the others because it can keep constitutes of reverse micelles phase in-variable to the most extent and keep proteins activity higher in the stripping aqueous phase, which is im-portant for the recycle of the reverse micelles system. Therefore, efficient back extraction method is impor-tant for the commercial scale application of reverse

micelles in the separation and purification of proteins.

2.2 New surfactants designed (synthesized) for reverse micelles

Despite their popularity as a research model sys-tem, AOT reverse micelles have some problems for protein solubilization such as variety limitation of protein extracted, protein denaturation in some of the extraction processes due to the strong electrostatic interactions, poor ability to release the proteins into aqueous medium during back extraction, and slow phase separation [25]. Therefore, a number of surfac-tants designed (synthesized) studies to form novel reverse micelles have been carried out to meet various research demands [26, 27].

In conjunction with the new surfactants designed (synthesized) for reverse micelles, the noteworthy articles were presented by Goto et al. [28, 29], who designed a number of synthesized surfactants such as di(tridecyl) phosphoric acid (DTDPA) and dioleyl phosphoric acid (DOLPA) for potential use in reverse micelles protein extraction. The new reverse micelles can easily extract proteins such as hemoglobin, which cannot be extracted by AOT reverse micelles. It is concluded that a suitable surfactant for protein extrac-tion must have high hydrophobic alkyl chains and should have a branched or unsaturated group to give a high solubility in aliphatic solvents, and its hydropho-bic structure should pack closely on the surface of the protein that offer steric hindrance to close packing. These researches develop the novel and perfect re-verse micellar systems for proteins extraction and in-dicate the structure characterization of the surfactant molecules, which can form reverse micelles at the early stage.

3 NONIONIC SURFACTANT-BASED REVERSE MICELLES

With the development of protein extraction by ionic reverse micelles, protein deactivation caused by the strong electrostatic interaction occasionally occurs in extraction process [25]. Therefore, some nonionic surfactants are used to form nonionic reverse micelles for proteins extraction to avoid protein deactivation. Tween 85 was usually used for reverse micelles ex-traction. It is proved that Tween 85 does not have a detrimental effect on the structure, function, and sta-bility of proteins solubilized in reverse micelles [30]. Naoe et al. [31, 32] successively developed Span 60/isopropanol/hexane and sugar ester DK-F-110/ isopropanol/hexane reverse micelles to extract cyto-chrome c and lysozyme. It is shown that the protein extractions using the two nonionic reverse micellar systems are both dependent on initial pH and buffer concentration, which indicate the existence of the weak electrostatic interaction in nonionic reverse micelles extraction. To obtain a biocompatible reverse micellar system without alcohols and saturated hydrocarbon solvents, reverse micelles composed of phospholipids

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as the biocompatible amphiphilic molecules and fatty acid or fatty acid esters as the biocompatible organic solvents were formed by Ichikawa et al. [33, 34] in which the maximal W0 in the organic phase and the size of the reverse micelles can be changed by con-trolling the salt concentration in the conjugated aque-ous phase. Wang et al. [35] applied the polyoxyal-kylene block copolymers/1-octanol/p-xylene reverse micelles to separate amino acids, and the driving force for the extraction process is verified to be mainly hy-drophobic and hydrogen-bonding interactions. An-other article also reported that the interactions be-tween proteins and TX-100 as a nonionic surfactant are hydrophobic and polar interactions [36]. It is con-cluded that the strong electrostatic interaction as ionic reverse micelles extraction is lacking in nonionic re-verse micelles extraction process. Therefore, the ef-fectiveness of protein separation by nonionic reverse micelles is limited, and the investigation on practical proteins separated by nonionic reverse micelles is re-ported scarcely due to the low extraction yield and selectivity [33].

4 MIXED REVERSE MICELLES

To solve the problems of the protein deactivation in ionic reverse micelles and the low extraction rate and selectivity of protein in nonionic reverse micelles, several attempts have been initially made to relieve the strong electrostatic interaction in ionic reverse micelles by adding nonionic surfactants to ionic re-verse micelles for protein solubilization, so various mixed reverse micelles emerge to solubilize proteins. The nonionic surfactant of Tween series such as Tween 80 were chosen to be added to AOT reverse micelles for enzymes solubilization [37], the enzy-matic activity in the mixed reverse micelles can be enhanced due to the adjustment of the microenviron-mental polarity surrounding the solubilized enzyme molecules by adding Tween 80. In addition, some other nonionic surfactants or ionic surfactants are used to form the mixed reverse micelles such as AOT-DOLPA [38], AOT-OPE4 [39], and CTAB-TRPO [19] reverse micelles for protein extraction and separa-tion. The results of extraction indicate that the mixed reverse micelles are more suitable for extracting and separating protein in both forward extraction and backward extraction than the single ionic reverse mi-celles. Besides, the strong electrostatic interaction is weakened by the other surfactant addition, and another important reason is that the size of the mixed reverse micelles increased with the introduction of the other surfactant molecules [40]. Therefore, a novel mixed reverse micellar system emerged by adding some bile salts such as NaTC, CHAPS, and SC [41 43] to the ionic reverse micelles in which the bile salts partici-pate to form the reverse micelles structure and in-crease the W0 and the size of the reverse micelles. Moreover, the bile salt concentration is the key factor that can affect enzyme solubility and activity in the mixed reverse micellar system [42]. However, the mixed reverse micelles cannot be used extensively in

biotechnology due to the complex system and limited applicability until now.

5 AFFINITY-BASED REVERSE MICELLES 5.1 Extraction and separation of affinity-based reverse micelles

Affinity-based separation techniques have exqui-site selectivity and have been used to purify bio-molecules. In recent years, affinity-based reverse mi-celles extraction and separation (ARMES) have been investigated for the separation of proteins with high selectivity and purification factor. This technique in-volves the affinity interaction between proteins and their affinity ligands are introduced into reverse mi-celles, which is the main driving interaction in extrac-tion process. The affinity interaction is divided into two fundamentally distinct modalities by the affinity ligands: (i) specific ligands and (ii) group ligands. For the former case, one affinity ligand has a very narrow specificity for a single compound (or a limited number of related compounds). Affinity-based reverse micelles extraction and separation technology is based on the biospecific association of molecules such as antibody- antigen. Adachi et al. [44] selectively separated chy-motrypsinogen using antichymotrypsinogen-antibodies as affinity ligands immobilized by covalently com-bining cholesteryl groups in reverse micellar system composed of tetra-oxyethylenemonodecylether. It is advantageous to use this system because it is highly selective and can be used for the protein if its antibody is available. However, antibody ligand is expensive and its instability may limit its application. In contrast, the group-specific interactions are well known for most biomolecules, which indicates one affinity ligand can bind multiform biomolecules. Therefore, group- specific ligands have been introduced into reverse micellar systems. These ligands include Cibacron Blue 3GA introduced by electrostatic interaction into CTAB reverse micellar system [45] or covalently im-mobilized to reversed micelles composed of soybean lecithin by a two-phase reaction [46], concanavalin A that can selectively bind soyabean peroxidase at pH 8 [47], the metal chelating ligands that can be used to extract hemoglobin as a hydrophilic head of the affin-ity cosurfactant [48].

Affinity-based reverse micelles extraction and separation show promising aspects of enhancing the selectivity, as well as the capacity of reverse micelles extraction, by introducing the affinity ligands. How-ever, the main surfactants used in these systems are still ionic surfactant such as AOT. The strong electro-static interactions between the ionic surfactants and proteins impede the affinity effect mostly under usual extractive conditions. That is, the selectivity increase is hindered by the strong electrostatic interactions un-der normal extractive conditions. Therefore, incorpo-rating affinity ligands into reverse micelles of non-ionic surfactants was proposed [44, 49]. Because the reverse micelles composed of nonionic surfactants only have a small ability to extract proteins [44, 48], introduction of an affinity ligand to the system can solubilize the desired protein only by the affinity

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interaction. However, the known nonionic surfactants are limited to form reverse micelles and are difficult to be separated from clear interface, so it is still a key problem to develop new nonionic surfactants, which can form reverse micelles with good phase-separation properties and high solubilizing capacities after intro-ducing affinity ligands.

5.2 Novel reverse micelles of Span 85 modified with Cibacron Blue F3G-A

We have recently developed a novel affinity-based reverse micellar system consisting of nonionic surfac-tant sorbitan trioleate (Span 85) modified with Ci-bacron Blue F-3GA (CB) and characterized the system for lysozyme solubilization [50]. It is shown that hy-drodynamic radius and W0 of the reverse micelles are significantly increased by the introduction of CB ligands (CB-Span 85 conjugate). Moreover, the exten-sive studies of lysozyme extraction and recovery in the CB-Span 85 reverse micelles indicate that the ex-traction is based upon the affinity interactions between lysozyme molecules and the CB ligands. We have en-hanced the solubilization capacity of the reverse mi-celles by adding hexanol to the system [51]. The schematic structure diagram of the CB-Span 85 re-verse micelles with hexanol addition is shown in Fig. 1, which is consisted with the variety of the hydrody-namic radius, aggregation number distribution, and W0 of the reverse micelles with hexanol addition. Fol-lowing is the further study on the extraction behavior of different proteins (lysozyme and ovalbumin) in the CB-Span 85 reverse micellar system with hexanol addition, and lysozyme is purified from a crude

chicken egg white solution with high activity rates and purification factors [6]. The recycling of the CB-Span 85 reverse micelles is also carried out for lysozyme purification, which exhibits good reusing capability of this micellar system.

Subsequently, the CB-Span 85 reverse micellar system has been applied as a protein refolding me-dium [9]. Under the optimized operating conditions of pH and the concentrations of urea and redox reagents, complete renaturation of lysozyme at 3 3.5 mg·ml 1 is achieved [9]. Furthermore, the artificial chaperones composed of CTAB and -cyclodextrin increased the refolding yield in a wide range of urea concentrations [52]. Another application of the CB-Span 85 reverse micellar system is used for Candida rugosa lipase (CRL) solubilization, and the system was character-ized and evaluated by using CRL-catalyzed hydrolysis of olive oil as a model reaction [4]. In summary, the affinity-based reverse micelles of CB-Span 85 system is potential and promising in biotechnology research, which is verified to be biocompatible and effective for protein solubilization in protein extraction, protein refolding, and enzyme catalyzed.

6 APPLICATION

6.1 Application in protein separation

Reverse micelles extraction has been developed from model proteins separation to practical protein system purification. Moreover, the latter application of reverse micelles attracted more and more attention of many scholars in recent years. Table 1 lists the appli-cations of various (ionic, mixed, affinity-based) re-verse micelles systems in practical protein system pu-rification. As shown in Table 1, most of the separated protein can be recovered by various reverse micelles. AOT reverse micelles system has been used exten-sively in recent years due to its larger water-pools and stable phase. However, the purification factors of the separated proteins by AOT reverse micelles are lower universally than the purification factors by affinity- based reverse micelles, which is caused by the interac-tion of protein and the reverse micelles. The ionic re-verse micelles extract protein mainly by electrostatic interaction and steric hindrance interaction [53], so its selectivity is low. However, the affinity-based reverse

Table 1 Some recent reports on reverse micelles for protein separation

Reverse micelles Target protein Source Recovery /% Purification factor Ref.

AOT nattokinase 0.7 mg·ml 1 (protein) fermentation broth 80 2.7 [18]

AOT soy protein 30 mg·ml 1 soy flour 60 [20]

AOT IgG 4.0 mg·ml 1 colostral whey 90 [53]

CTAB arginine deiminase 30 mg·ml 1 crude enzyme 85 4.52 [54]

CTAB-TRPO lipase 2.0 mg·ml 1 industry lipase 70 [19] C10E4 immobilized anti-CTN

antibodies chymotryp-sinogen 7×10 6 mol·L 1 mixed proteins 10.8 [44]

CB-Span 85 lysozyme 5.0 mg·ml-1 crude chicken ovalbumin 71 21.2 [6] Recovery denotes the total yield of the separated protein, i.e., the product of forward extraction yield and back extraction yield. Purification factor is equal to the protein activity in stripping solution divided by protein activity in initial feedstock.

Figure 1 Schematic structure of the CB-Span 85 reverse micelles with hexanol addition

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micelles extract protein by affinity interaction and its selectivity increases significantly. Therefore, the affinity-based reverse micelles system can enrich and purify protein efficiently.

Compared with other protein separated tech-niques such as chromatography, membrane separation, and electrophoresis, reverse micelles extraction is liquid-liquid extraction, which is cost-effective, easily scaled-up, and continuous operation for whole broth processing. However, there is a major problem with emulsion formation when real broths are separated by reverse micelles [55]. The effort of two main demulsi-fication methods on reverse micelles extraction has been carried out. One is by adding a demulsifier such as nonionic surfactant, the other is by developing ex-traction equipment, which can avoid the formation of stable emulsions in extraction process.

6.2 Application in protein solubilization and re-folding

The production of proteins via genetic engineer-ing often forms precipitating inclusion bodies with incorrect folding of the proteins. Thus, a protein re-folding step is essential to regain the biological activ-ity of these denatured proteins during downstream processing. However, the conventional dilution re-folding process usually results in very low yield of the low-concentration ( 1 mg·ml 1) protein recovery. In order to solve this problem, many other refolding techniques such as fed-batch refolding, molecular chaperones assisted refolding, refolding chromatog-raphy, and reverse micelles refolding have been stud-ied. Reverse micelles are utilized to accommodate unfolded proteins as the refolding medium. Because the size of the water pools is comparable to the size of proteins and can be controlled by adjusting the W0 of the micelles, protein molecules can be isolated from each other during the refolding process. Consequently, complete renaturation of high-concentration proteins can be obtained by reverse micelles at optimized con-ditions [56 59]. Unlike other refolding techniques, re-verse micelles refolding is believed to be a synchro-nous technology for the separation and refolding of proteins in downstream processing with its simple operation and low cost. However, reverse micelles

refolding is limited in the proteins with low molecular weight due to the small size of the reverse micelles ( 10 nm).

Nowadays, the familiar methods of protein re-folding by reverse micelles include liquid-liquid method [8] and solid-liquid method [56, 57], which are divided according to the different states of protein solubilized in and recovered from reverse micelles. Table 2 lists several recent reports on protein solubili-zation and refolding by reverse micelles. As shown in Table 2, AOT reverse micellar system is extensively used in protein refolding. Goto et al. successfully dealt with the high concentration of 4.8 mg·ml 1 ri-bonuclease A complete refolding at the optimum con-ditions in AOT reverse micelles by solid-liquid method [57]. Furthermore, other protein refolding methods such as dialysis [8] and adding the molecular chaperone (GroEL) [60] were introduced to reverse micelles refolding of protein, and this obviously im-proved the refolding effect in continuous operation and the refolding rate, respectively. However, due to the strong interactions between the surfactant and the proteins, the renatured proteins solubilized in AOT reverse micelles are difficult to be stripped to an aqueous solution and have to be recovered by precipi-tation by adding cold acetone [8, 57, 60]. Moreover, not all the proteins can be refolded by the AOT reverse micelles because some proteins such as carbonic an-hydrase B form precipitates by the strong interaction with AOT molecules [61]. Hence, nonionic surfactant- based reverse micelles have been developed for pro-tein refolding [9, 52, 61]. These micellar systems are advantageous in the mild condition for protein refold-ing, so the renatured proteins can be recovered easily using a stripping solution of high ionic strength with-out unfavorable interaction of ionic reverse micelles.

7 CONCLUSIONS

This article has indicated that surfactant that forms reverse micelles in organic solvents is the key element for useful reverse micelles for application in protein separation and refolding. The drawbacks of ionic surfactant-based reverse micelles such as protein denaturation and poor ability to release the proteins in back extraction mainly due to the strong electrostatic

Table 2 Some recent reports on reverse micelles for protein refolding

Reverse micelles Protein Refolding concentration/mg·ml 1

Renaturation yield/% Recovery method Ref.

AOT RNase A 0.5 100 [58]

AOT RNase A 1.0 5.0 75 100 [56]

AOT RNase A 2 100 precipitated with acetone [8]

AOT RNase A 4.8 100 precipitated with acetone [57]

AOT RNase A (inclusion body) 0.4 100 precipitated with acetone [60]

AOT galactase oxidase (inclusion body) 0.15 precipitated with acetone [59]

tetraethylene glycol dodecyl ether CAB 0.1 70 1mol·L 1 KCl stripped [61]

CB-Span 85 lysozyme 3 3.5 100 1mol·L 1 MgCl2 stripped [9]

CB-Span 85 lysozyme 3.5 5.9 70 100 1mol·L 1 MgCl2 stripped [52]

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interactions have been clearly recognized. Hence, nonionic surfactant-based reverse micelles have been studied to solve these problems. This also resulted in some new problems such as low extraction yield and selectivity. As a result, mixed reverse micelles with ionic and nonionic surfactants have been developed to decrease the protein deactivation in ionic reverse mi-celles and to increase the extraction yield and selectiv-ity of proteins. Furthermore, affinity-based reverse micelles composed of nonionic surfactant coupled with affinity ligands have been investigated for the separation of proteins with high selectivity and purifi-cation factor, which can provide a mild microenvi-ronment and keep high activity of proteins. It is con-sidered that affinity-based reverse micelles have a great potential for extensive application in biotech-nology. Nowadays, the upstream gene cloning tech-niques and downstream protein purification operations are tied in by the development of recombinant DNA techniques. Expression vectors have been developed to express fusion proteins with a known sequence (e.g., polyhistidine) that can bind specifically to an affinity ligand (e.g., transition metals). Thus, the expressed protein products can be easily captured by a highly specific affinity separation (e.g., metal chelate bind-ing). Hence, new affinity-based reverse micellar sys-tem is demanded for wide applications in protein separation and refolding in biotechnology. A suitable nonionic surfactant coupled with metal-chelate ligand is proposed to form this kind of novel reverse micelles. This system can be widely used to solubilize the histidine-tagged fusion proteins and can be applied to separate and refold proteins in a single step.

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