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 (刘杨), DONG Xiaoyan (董晓燕) and SUN Yan (孙彦)**
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 In the past three decades, the application of reverse

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-
－ －
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-

meter size ＜100 nm), large surface area (102-103 lization in reverse micelles. It is also generally recog-
m2·cm 3), viscosity comparable with pure organic nized that few current surfactants can form reverse
－

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].
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
950 Chin. J. Chem. Eng., Vol. 16, No. 6, December 2008
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
 Chin. J. Chem. Eng., Vol. 16, No. 6, December 2008
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
951
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
952 Chin. J. Chem. Eng., Vol. 16, No. 6, December 2008
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
Figure 1 Schematic structure of the CB-Span 85 reverse
micelles with hexanol addition
Table 1 Some recent reports on reverse micelles for protein separation
Reverse micelles Target protein
－1
AOT nattokinase 0.7 mg·ml
AOT soy protein 30 mg·ml
AOT IgG 4.0 mg·ml
CTAB arginine deiminase 30 mg·ml
CTAB-TRPO lipase 2.0 mg·ml
－6
C10E4 immobilized anti-CTN chymotryp-sinogen 7×10
antibodies
CB-Span 85 lysozyme 5.0 mg·ml-1 crude chicken ovalbumin
① Recovery denotes the total yield of the separated protein, i.e., the product of forward extraction yield and back extraction yield.
② Purification factor is equal to the protein activity in stripping solution divided by protein activity in initial feedstock.
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 β-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
① ②
Source Recovery /% Purification factor Ref.
(protein) fermentation broth 80 2.7 [18]
－1
soy flour 60 - [20]
－1
colostral whey 90 - [53]
－1
crude enzyme 85 4.52 [54]
－1
industry lipase 70 - [19]
－1
mol·L mixed proteins - 10.8 [44]
71 21.2 [6]
 Chin. J. Chem. Eng., Vol. 16, No. 6, December 2008 953

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

Refolding concentration/ Renaturation
Reverse micelles Protein Recovery method Ref.
mg·ml 1
－
yield/%
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]
－1
tetraethylene glycol dodecyl ether CAB 0.1 ＞70 1mol·L KCl stripped [61]
－1
CB-Span 85 lysozyme 3-3.5 100 1mol·L MgCl2 stripped [9]
－1
CB-Span 85 lysozyme 3.5-5.9 70-100 1mol·L MgCl2 stripped [52]
954 Chin. J. Chem. Eng., Vol. 16, No. 6, December 2008
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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