A Review: Molecular Chaperone-Mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins

Cell Biochemistry and Biophysics
https://doi.org/10.1007/s12013-021-00970-5
REVIEW PAPER
A Review: Molecular Chaperone-mediated Folding, Unfolding and

Disaggregation of Expressed Recombinant Proteins
Komal Fatima1 Fatima Naqvi1 Hooria Younas
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1
Received: 22 July 2020 / Accepted: 1 February 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active
proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk.
E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation,
simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes
with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding.
Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one
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major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular
chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones
have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli.
Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as
trigger factor, GroEL, DnaK and ClpB.
Keywords Chaperones Inclusion bodies Trigger factor GroEL/GroES DnaK/DnaJ/GrpE ClpB
● ● ● ● ●
Introduction handle genetic modifications due to convenient selection of

mutants. Furthermore, E. coli can efficiently take up foreign
Recombinant DNA technology is employed to generate new DNA incorporated into a plasmid in consort with a higher
genetic combinations utilizing genomic fragments of dif- rate of recombinant protein expression [2]. Almost 86% of
ferent organisms. In 1973, Stanley Cohen and Herbert the three-dimensional protein structures that have been
Boyer, pioneers of genetic engineering, combined the submitted into the protein data bank (PDB) to date, have
genetic information from two different plasmids to con- been expressed in E. coli [3]. The first commercialized
struct a new recombinant plasmid with functional and human protein by Genentech, somatostatin, was genetically
structural characteristics of both parent genomes [1]. Mul- amended and expressed under the E. coli expression
tiple hosts which include yeast, bacteria, animals, and plants machinery [2]. So far, it is not an easy task to attain
have been used for recombinant protein production fol- heterologous gene expression in E. coli due to multiple
lowing basic principles of genetic engineering. Among drawbacks. Such as, the inability to attain posttranslational
these, Escherichia coli is considered as the most desirable modifications of proteins, methionine cleavage from amino-
host for recombinant protein production from eukaryotic or terminal, complex disulfide bond formation and insoluble
prokaryotic origin due to various reasons. E. coli has a short protein expression which leads to the formation of inclusion
generation period of nearly 20 min so it can be easily cul- bodies (IBs) or insoluble aggregates [4].
tured using inexpensive growth media and it is easier to Inclusion bodies are inactive, misfolded protein aggre-
gates (ranging in size from 50 to 800 nm) which form due to
improper intra- or intermolecular interactions [5]. Protein
aggregates are non-native secondary structures and are
* Hooria Younas localized to intracellular environments due to poor solubi-
hooria.younas@kinnaird.edu.pk
lity in aqueous medium [6]. Apart from protein aggregates
1
Department of Biochemistry, Kinnaird College for Women, being nonspecifically-bound disordered precipitates of
Lahore, Punjab, Pakistan polypeptide chains, they have also been reported to emerge
from specific interactions between partially folded inter- hydrochloride which disrupt the improper intermolecular
mediates [7]. This interaction specificity might be derived interactions [20].
from previously formed aggregates that directs the deposi- E. coli has developed a complex protection system to
tion of monomers at a single point [8]. Thus, IBs are promote the refolding of mis-folded proteins and prevent the
regarded as intracellular foci (cellular component which is aggregation of nascent peptides [21]. Molecular chaperones,
different from surrounding elements) in which protein also known as heat shock proteins (HSPs), are folding mod-
aggregates accumulate [5]. They are usually hydrated ulators of ubiquitous class of proteins [13]. HSPs maintain
sponge proteins with an outer surface covered by hydro- cellular homeostasis under favorable and unfavorable growth
phobic residues [9] which are abnormally exposed to the conditions by inhibiting the aggregation of poorly folded
cellular content causing the formation of inactive IBs [10]. proteins, in both prokaryotic and eukaryotic cells [21–23].
In E. coli, IBs are formed due to high rate of protein They are also involved in the regulation of protein degrada-
expression [11]. The expression of a foreign gene in E. coli tion by assisting lysosomal or autophagy mechanisms [24].
is influenced by several factors, including the secondary Molecular chaperones are sub-divided into three main
structure of mRNA, protein folding, use of codons, the families subjected to their mode of action. These include
extent of the toxicity and posttranslational modifications of holding chaperones (holdases), folding chaperones (foldases),
the foreign gene [12]. In this regard, IB formation is and disaggregating chaperones (disaggregases). Holdases
attributed as a major shortcoming for increasing the yield of function in the absence of ATP to stabilize proteins that are
recombinant proteins in research or pharmacological partially folded by functioning in concert with foldases [25].
industries [13]. High-quality proteins in adequate amount Holdases include inclusion body associated protein B (IbpB)
are the requisite for protein structural, functional, and bio- [26] and trigger factor (TF) [13]. Foldases, such as, growth
chemical analysis. Despite the lack of any universal strategy essential large protein (GroEL) and DnaK, facilitate the
to resolve the issues concerning recombinant protein pro- refolding or unfolding of improperly folded proteins through
duction in E. coli, certain methods have been devised to ATP-driven reactions [13]. Stringent protein aggregates are
improve protein soluble expression as different proteins solubilized by disaggregases such as ClpB (caseinolytic
behave differently under various conditions [14]. These peptidase protein B) [27]. The basic features of prokaryotic
include varying the host strain, vector [14], amount of chaperones are discussed in Table 1. Chaperones do not
inducers [15], temperature [16], promoters [17], fusion tags possess any information regarding the stereo-chemical prop-
[18] and co-expression with other genes such as molecular erties of the target protein that can help to obtain correct
chaperones [19]. IB solubility is mostly achieved through folding. Proper protein folding is ensured by preventing or
stimulants like hydrochloric acid, urea and guanidine correcting the improper folding interactions between
Table 1 Major prokaryotic (E. coli) chaperones with their structural and functional properties
Chaperones General Substrate Specificity Structural Functional Properties References
Characteristics Features
• Trigger Factor Class: Holding Stretch of eight Aromatic amino Three ATP-Independent chaperone bound at [170]
Chaperone. acids in the substrate. Domains: ribosomal exit site. First interaction of
Size: 48 kDa. 1. N-Terminal nascent polypeptide.
2. Middle
3. C-Terminal
• GroEL Class: Folding Loop-like hydrophobic patches Three GroEL mediates folding of misfolded [171]
Chaperone. of substrate residues. Domains: proteins by enclosing them in a cage in
Size: 60 kDa. 1. Equatorial the presence of GroES and ATP.
2. Intermediate
3. Apical
• DnaK Class: Folding Hydrophobic residues on Two domains: ATP-dependent chaperone that [172]
chaperone substrate peptide. 1. N-terminal functions in collaboration with DnaJ
Size: 70 kDa 2. C-terminal and GrpE to properly fold unfolded
proteins.
• ClpB Class: Exhibits different substrate Three domains: ATP-dependent chaperone that [173]
Disaggregating specificity by considering the 1. N-terminal functions with DnaK/J system to
chaperone general surface properties of 2. AAA+ disaggregate protein aggregates by
Size: 95 kDa substrate peptides. domain 1 extracting single polypeptides through
3. AAA+ its central pore.
domain 2
polypeptides [10]. Chaperones identify improperly folded (TF55) and archaebacteria (thermosomes). Type II chaper-
proteins by their anomaly exposed hydrophobic residues that onins do not require co-chaperones (such as GroES) to
would ideally be buried within the protein core under normal function as some part of the chaperone structure can act as a
conditions. Stabilization against protein aggregation is replacement for the co-chaperone [34, 35]. This group of
achieved by the substrate-chaperone binding [13]. chaperones can be understood using the eukaryotic CCT.
There is subtle degree of selectivity present in substrate Since type II chaperonins do not function in coordination
capture due to some differences present in the recognition with co-chaperones, protrusions on the tip of the apical
sequences of substrate proteins. Chaperones typically target a domain in every CCT subunit act as a lid for the central
short stretch of hydrophobic residues flanked by basic amino cavity [36–38]. The equatorial domain region of type I and
acids. Chaperones release the bound substrate after governing II chaperonins shows significant homology and major dif-
conformational changes by ATP hydrolysis if proper folding ferences arise due to difference in the sequence of the
has taken place, otherwise the substrate binds to the chaper- central region [39]. The differences between type I and II
one again until the native conformation is attained [21]. chaperonins can be understood by comparing GroEL and
Molecular chaperones are found in the cytoplasm and peri- CCT. CCT has higher complexity and substrate specificity
plasm of prokaryotes and in cytoplasmic organelles such as than GroEL [39]. This is due to its role in facilitating proper
mitochondria, endoplasmic reticulum, chloroplast and nucleus folding of major cytoskeleton proteins such as luciferase,
of eukaryotes [28]. During heterologous protein synthesis in tubulin, actin, cyclin E and Gα-transducin [40].
E. coli, protein aggregation is frequently observed which A ring structure with a hollow cavity is a common fea-
further leads to formation of IBs in cells and this problem is ture of all chaperones, however, the conformational changes
mainly administered through molecular chaperones in cell in the chaperones due to ATP attachment and detachment in
[29]. The main conserved chaperones which are responsible a reaction cycle differ between prokaryotes and eukaryotes
for de novo protein refolding in E. coli are trigger factor (TF), [41]. For example, in the prokaryotic GroEL/GroES cha-
GroEL/GroES, DnaK/DnaJ/GrpE, and ClpB [30, 31]. This perone system, all subunits simultaneously undergo con-
review focuses on the basic structural features and mechanism formational changes [42, 43] however, the eukaryotic CCT
of action of major E. coli molecular chaperones and their role subunits undergo sequential conformational changes [44].
in proper folding of nascent polypeptides during over- This difference is mainly due to the difference in the type of
expression in E. coli. proteins that prokaryotic and eukaryotic chaperones facil-
itate [41]. Eukaryotic proteins are generally larger with
multiple domains, whereas prokaryotic proteins are com-
Comparison between Prokaryotic and paratively shorter with a single domain.
Eukaryotic Chaperones Another example that highlights the difference between
prokaryotic and eukaryotic chaperones is the difference in
Protein folding is crucial for an ideal protein structure- the hydrolytic and nucleotide-binding proteins of DnaK and
function relationship in all domains of life such as prokar- Hsp70 chaperones [45]. This is due to the absence of a
yotes, archaea, and eukaryotes. Despite the protein structure nucleotide exchange factor like GrpE in eukaryotes. For the
being already decided by the amino acid sequence, various DnaK reaction cycle, ADP release is the rate-limiting step
factors influence the folding process. Therefore, to ensure [46] and the presence of DnaJ and GrpE is important for
the proper folding of proteins, molecular chaperones are maximum ATPase activity [45]. Whereas for the Hsp70
required. However, not all proteins such as the insect apo- reaction cycle, the rate-limiting step is ATP hydrolysis and
lipophorin III (apoLp-III) require the assistance of chaper- only Hsp40 stimulates ATPase activity [47, 48]. Prokar-
ones for proper folding due to the presence of an extra yotic chaperone systems are generally understood better due
α-helix which facilitates protein folding [32]. to them being simpler than eukaryotic chaperone systems.
Regardless of their origin, chaperones are classified in
two groups of chaperonins in terms of their degree of
homology [33]. The first group consists of chaperonins with Prokaryotic Chaperones
higher homology and originate from the bacterial cytosol
(GroEL), chloroplasts (Rubisco-binding protein) and mito- The conversion of genetic information from the genome to
chondria (hsp60). The GroEL/ES chaperone system (dis- three-dimensional proteins is a complex process. Polypep-
cussed later in the article) can serve as a model to tides cannot fold instantaneously into their native con-
understand the function of group I chaperonins. The second formations after being translated from ribosomes until the
group consists of chaperonins with a significant yet low complete synthesis and release of the polypeptide chain.
homology and are found in the cytosol of eukaryotes During the process of protein synthesis, newly synthesized
(chaperone containing TCP-1 (CCT)), thermophilic bacteria proteins are exceedingly prone to immature folding,
misfolding and aggregate formation, which is supplemented ribosomal-associated protein factors are present in eukaryotic
by formation of IBs. In the E. coli cytosol, molecular cha- and archaeal cells [56], whereas, in E. coli TF is the primary
perones play a key role in resolving this problem [21]. chaperone that interacts with the nascent proteins. TF is
Prokaryotic molecular chaperones such as trigger fac- abundantly found in the cytosol, with ~20,000 copies/cell
tor, DnaK/DnaJ/GrpE and GroEL/ES are involved in the and ~2–3 fold higher in molar concentration than ribosomes
folding of numerous cellular proteins [49]. Ribsome- [57]. Moreover, a fraction of trigger factor which is not
associated trigger factor interacts with newly synthesized bound to ribosomes is present in dimer form that is in
proteins emerging from the ribosomal exit site [50, 51]. equilibrium with its monomeric form.
Trigger factor prevents the formation of aggregates or IBs TF attaches with the larger ribosomal subunit near its exit
by holding the newly synthesized polypeptides for a site through its tail and the other two TF domains form a
limited time. This limitation however might result in the cradle that hangs along the ribosome [58]. TF dissociates
aggregation of multi-domain larger proteins. Therefore, from the ribosome after attaching with the protein and
such proteins are further transferred to the DnaK/DnaJ/ prevents its aggregation by associating with the folding
GrpE system which assists in the folding of misfolded chaperones, GroEL [59] and DnaK [52]. Precisely, not
proteins. Some proteins might skip through this system enough information is available regarding the specific
and are further transfered to other chaperones [52]. At this structural properties of the client substrates and how TF
stage, the GroEL/ES system contributes to the folding of affects their conformational dynamics.
misfolded proteins (Fig. 1). Some proteins are quite large Trigger factor (48 kDa) consists of 432 residues and is
and form stringent IBs, therefore they are subjected to comprised of three structural domains: the N-terminal
ClpB chaperone which disaggregates the IBs and unfolds domain (tail), middle domain (head) and the C-terminal
the proteins [53] (Table 1). These unfolded proteins are domain (arms), which adopt an elongated, unique and “dra-
again folded by the assistance of either DnaK/DnaJ/GrpE gon-like” structure upon folding [60] (Fig. 2a, b). The N-
or GroEL/ES [54]. terminal domain contains the amino acid residues 1 - 149 and
carries a motif sequence which is known as the TF-signature-
Trigger factor motif (FRK motif; Fyn-related kinase, tyrosine related kina-
ses) that mediates ribosomal docking (Fig. 2a, b) [61]. The
Trigger factor (TF) is the only ATP-independent and motif consists of a helix-loop-helix sequence and the residues
ribosome-associated protein present in E. coli [55]. Different Gly-Phe-Arg-X-Gly-X-X-Pro [62]. The N-terminal domain
Fig. 1 De novo general folding pathway for nascent polypeptides in through this pathway, and are subjected to GroEL (PDB ID: IAON
E. coli. Trigger factor (PDB ID: 1W26 [148]) binds with the ribosome [154]) and its co-chaperone GroES in the presence of ATP. Some
at its exit site and holds the nascent polyeptide chain to prevent its proteins that are too large to handle may form aggregates and so are
misfolding. If the polypeptide gets misfolded, it then transfers to the disaggregated by ClpB (PDB ID: 4D2X [155]). The native protein
DnaK/DnaJ system (PDB ID: 2KHO [152]) and to its cofactors (GrpE shown in the figure is a serine-aspartate repeat-containing protein
(PDB ID: 3A6M [153]) and ATP). Some proteins can also skip G (PDB ID: 5IEJ [146])
Fig. 2 Trigger factor structure and mechanism of action. a Three- exit site mainly through hydrophobic interactions. TF binding with the
dimensional structure: Trigger factor (PDB ID: 1W26 [60]) consists of ribosome persists for about 10 s after it remains attached to the elon-
three domains: the N-terminal domain (residues 1–144) in gray which gated nascent chain. Its displacement allows a second molecule to
assists in ribosomal binding, the PPIase domain (residues 145–247) in attach at the exit site and bind with the polypeptide. Simultaneously,
yellow which captures the polypeptide chain and the C-terminal after completing translation, the polypeptide may be folded into its
domain (residues 248–432) in red, present at the center containing native structure (PDB ID: 5NLU [166]) or may be transferred to
major substrate binding sites. b Trigger factor reaction cycle: Trigger downstream chaperone molecules
factor monomer binds with newly translated protein at the ribosomal
is responsible for TF binding with the ribosomal protein L23 in the chaperone activity to some extent by providing
that facilitates its chaperone activity [63]. The tail sequence is attachment to the auxiliary ribosomal binding site at the L29
between the residues 1–110 while the residues 111–149 form protein [68]. Moreover, in some cross-linking experiments
a long linker segment that joins the tail with the other PPIase domain has been found to aid in TF interaction with
domains [64]. The TF middle domain consists of the residues longer chains [69].
150–245 and it belongs to the FK506 binding protein The TF C-terminal domain (CTD) consists of residues
(FKBP) family because it possesses peptidyl-prolyl cis/trans 246–432, almost half of the protein sequence, due to which
isomerase (PPIase) activity [63]. The TF substrate region it is regarded as the largest domain and presents major
recognized by the PPIase domain contains a stretch of eight chaperone activity (Fig. 2a, b). The CTD is present in the
aromatic and basic amino acids [30]. It forms the head of the center of the structure and has two distended arms to
TF structure and is located at a distance from the ribosomal hold the nascent polypeptide chain [41]. A linker sequence
binding site [65] (Fig. 2a). The chaperone activity of TF is containing residues 111–133 connects the N-terminal
independent of the PPIase domain, making its presence in the domain with the PPIase domain and allows these domains
structure questionable [66, 67]. The PPIase domain may help to fold in a way that places the CTD in the middle of the 3D
structure [43]. It has been observed in a study by Zeng et al. despite the absence of the middle domain, TF exhibited
[70] that an intact CTD structure greatly helps in the wild-type chaperone activity and contained a major sub-
refolding of some denatured proteins and inhibits protein strate binding site at the N-terminal domain. The second
aggregation. The absence of the CTD can impair the TF model stated that TF protects the nascent polypeptides
chaperone activity. from degradation and aggregation, by providing it with a
TF accommodates its substrate in the entire inner cavity space for folding [56]. TF always interacts with its sub-
due to lack of a specific, single substrate binding site strates through hydrophobic interactions by inhibiting the
[70, 71]. The mixed hydrophobic and hydrophilic character immediate misfolding of the nascent chain that is in line
of TF assists in the interaction with the substrate. Con- with what the two models state [51]. Furthermore, the
tinuous patches of hydrophobic residues are also often attachment between the TF and its substrate is disrupted by
present [72]. By comparing the structural data of TF in free, the release of a nascent domain which is completely folded
substrate-bound, and ribosomal bound state, it has been in the TF cradle [56]. The kinetics of the mechanism of
postulated that TF is a “flexible molecule”. It has “hinge- action of TF with its substrates is yet to be fully understood.
bending” rotations for the CTD, “intradomain rotations” for
the N-terminal domain, and “interdomain flexion” for GroEL
the middle domain [68, 73]. These rotations help TF to
accommodate complex substrate flexibly, adapt different The large, barrel-shaped oligomeric proteins which play key
states either for ribosomal-bound or unbound state and roles in the folding and maintenance of cellular network of
expedite the co-existence of other ribosome-associated chaperones are known as chaperonins [75, 76]. The earliest
translation factors. oligomeric folding chaperones are GroEL and GroES,
Saio et al. showed that TF has four distinct binding sites which were discovered in 1970s in the E. coli cytoplasm as
that are distributed along its inner binding scaffold [74]. All an essential component for the λ-phage assembly [77].
four binding sites are not bound to the substrate altogether, These chaperones utilize the energy released from ATP
instead they bind in varying order. These binding sites form hydrolysis to catalyze the folding of proteins [78]. GroEL is
hydrophobic pockets of nonpolar residues that interact with known as the most dominant protein of E. coli that is found
the hydrophobic regions of 6–10 amino acids in the sub- to be in a significantly higher concentration under heat
strate protein [53]. Due to the flexibility of these binding shock conditions [79]. GroEL can interact with newly
sites, they can interact with a large, diverse, and unrelated synthesized proteins for assistance in folding and also with
polypeptide population. By using all four binding sites, TF those proteins that have lost their intact native structure
can interact with up to 50 substrate residues [74]. [80, 81]. Through proteomic studies, it has been shown that
The precise attachment of TF at the ribosome has been the chaperonin system is compulsory for the folding of
deduced by examining the three-dimensional crystal struc- ~80% proteins [82, 83] or more [76]. In addition to this,
ture of the complex formed by the TF N-terminal domain ~30% of the bacterial proteins are unable to fold into their
and 50S ribosomal subunit [60]. TF binds with the riboso- native conformation during the absence of GroEL [80].
mal protein L23 at the exit site and hunches over the nascent Interestingly, any kind of disturbance in the GroEL
polypeptide while exposing its inner hydrophobic residues expression level can lead to the death of bacterial cell [75].
[62]. The exposed TF hydrophobic cradle-like structure Although, there is no patent specificity of GroEL for its
provides a shielded environment to capture the newly syn- substrate proteins, but it roughly shows attachment with
thesized polypeptide emerging from the ribosomal exit site exposed hydrophobic stretches of proteins.
[50]. This prevents any improper folding, aggregation, and The crystallographic structure of GroEL (547 residues)
premature degradation of the proteins (Fig. 2c). was first determined in 1994 [84]. GroEL contains a
Based on the substrate type, two models are proposed for cylindrical, thick-walled, porous structure with a higher
describing the fate of the nascent polypeptide. According to length than width and has a central cavity to enclose its
the first model, TF constrains the nascent chain inside its target protein (Fig. 3). The GroEL complex has two hep-
cradle and allows the formation of a properly folded protein tameric rings consisting of ~57kDa subunits, arranged in a
till the genetic information is fully translated. In support of cylindrical manner [85]. Each subunit has three domains of
this model, it has been observed through crystallographic discontinuously arranged sequence elements, such as, the
studies that TF can only accommodate a polypeptide of equatorial domain comprises of residues 6–133 (E1) and
15kDa. A typical domain size of substrate polypeptide 409–523 (E2). An equatorial domain is a highly well-
ranges from 100 residues (11kDa) to 130 residues (14kDa). ordered and helical domain which forms a solid foundation
This makes the TF cradle ideal for co-translating multi- at the baseline of the GroEL ring. It provides inter-subunit
domain proteins. This statement was found to be in line contacts and mostly comprises of those residues that are
with the study of Kramer et al. [50], that reported that involved in the ATPase activity [86]. There are 23 residues
present in the equatorial domain which protrude into the acids Gly-Gly-Met which protrude into the central cavity of
inner cavity while occluding the passage in the cavity. the crystal structure. All the equatorial domain residues in
These residues have repeated sequences of three amino the GroEL structure are responsible for inter- and intra-ring
Fig. 3 Structure and mechanism of action of the GroEL/ES complex. long loop with the sequence residues E16–A32. These
a, b cis & trans (PDB ID:1AON Chain A and H) ring conformations residues were visualized only in one of the barrels of the
of the GroEL single subunit. c The GroEL single subunit consists of
GroES standalone structure but are seen in all barrels when
three domains: the apical domain (olive) which entraps the substrate
and interacts with GroES, the intermediate domain (I1; teal & I2; present in complexed form [74]. It has been observed
smudge) that is responsible for conformational flexibility of the GroEL through an NMR study, that these residues become more
protein and the equatorial domain (E1; dark salmon & E2 light blue) structured upon interaction with GroEL due to which it can
that aids in the attachment of the two heptameric rings and possesses
be said that they form an interface between GroES and
the nucleotide binding site. d GroEL/ES complex: GroEL/ES subunits
interact with each other through the equatorial domain and with GroES GroEL [80]. GroES associates with GroEL by serving as a
through apical domain. e Mechanism of action: A bullet-cycle model small lid for the GroEL central cavity [88]. The GroEL/ES
for GroEL mode of action. GroEL cis conformation ring binds with the complex mainly contains aliphatic amino acid side chains
misfolded protein and seven molecules of ATP. Later, GroES binds
consisting of GroES I25, V26 and L27 and GroEL L234,
with the apical domain which confers structural changes in the GroEL
ring which converts into trans conformation. After ATP hydrolysis, L237 and V264 [89].
the misfolded protein gets entrapped in the GroEL/ES mini cage. GroEL was found to interact with a wide range of
When another protein and seven ATPs bind to the GroEL complex, the denatured proteins in vitro, while it has shown binding with
formerly folded polypeptide is released, hence completing the cycle.
~250 proteins under in vivo conditions and ~20 or 67%
GroEL and GroES single subunits have been drawn from Swissmodel
and their subsequent chains were downloaded by Pymol (Chain A, H proteins are from cytosolic origin that are essential to cell
and O of 1AON) [76]. Typically, an open ring of GroEL captures a single
misfolded polypeptide per ring and assists in its refolding
and prevents it from aggregation [73, 84]. The GroEL
interactions between the subunits (Fig. 3a, b). The equa- hydrophobic residues located on the helices H and I are
torial domain also holds two separate rings in a back-to- shown to interact with substrate polypeptides [73, 82].
back staggered manner and each subunit in one ring inter- The mechanism of action for GroEL and its co-chaper-
acts with two other subunits in the other ring [85]. one, GroES, has been illustrated in Fig. 3. The cycle is
The apical domain (A; 233–267 residues) is present at directed by the asymmetric activity of two GroEL hepta-
the end terminal of the central cavity. It shows less order meric rings following the attachment of ATP molecules
and flexibility in its structure which blocks hinge move- [85]. There is a cooperative binding of seven ATP mole-
ments around the link that connects it with the intermediate cules to each subunit of the GroEL ring at their equatorial
domain [87]. These domains are present at the entrance of domains [86]. Upon ATP binding, small changes are
the ring cavity that are attached to the equatorial domains induced in the direction of both the apical and intermediate
via the intermediate domains [85] (Fig. 3a–b). The apical domains. This converts a GroEL molecule from trans to cis
domain exposes two helices named as H (233–243 residues) conformation allowing for the feasible attachment of
and I (255–267 residues) that have hydrophobic residues GroES. After the attachment of GroES to the GroEL ring,
protruding into the inner cavity. These hydrophobic resi- further changes take place in the orientation of both
dues are responsible for the first line interaction of substrate domains. The GroEL ring undergoes an elevation of 60°
proteins with the GroEL ring [86]. The intermediate domain and a 90° twist in clockwise direction at G192 and G395
(I1; 138–192 and I2; 376–410 residues) is a small, slender residues in the intermediate domain [74, 87]. This forms a
peripheral structure of the ring which connects the apical true GroEL/ES complex which has now changed the
and equatorial domains. This domain is responsible for the hydrophobic cavity to a hydrophilic cavity [77].
structural changes in the entire protein upon the binding of Following these variations, the misfolded substrate
the substrate and nucleotide molecules (ATP) [85]. protein that was formerly entrapped in the GroEL cavity
due to hydrophobic interactions gets rapidly exposed to
GroES the hydrophilic space of GroEL/ES cavity. This interac-
tion facilitates the folding of misfolded protein [90]. After
GroES is a heptameric ring that consists of seven identical encapsulation of the protein into the GroEL cavity, fold-
beta-barrel composed subunits. The protein is folded into a ing proceeds, which is the longest part of the cycle
single domain consisting of nine beta-strands [77]. A (around 8–10 s) [91]. When this stage ends, ATP mole-
mobile, hydrophobic, flexible loop of 22 amino acids is cules convert into ADP molecules facilitating the release
present in between the second and third beta-strand which of the ligands, GroES and ADP by weakening very strong
extends from each β-barrel when it is present in isolated and stable interactions with GroEL [86]. The release of
form. Through this loop, GroES forms a stable association ligands from the GroEL ring is physiologically triggered
with the GroEL apical domain by interacting with the H and by the cooperative binding of ATP and additionally the
I helices (Fig. 3) [78, 79]. GroES standalone ring has a very binding of another substrate protein in the opposite ring
similar structure to its ring when in complex except for a [91] (Fig. 3e).
ATP hydrolysis in one round of the chaperonin cycle DnaK is an ATP-dependent chaperone which forms a
provides the direction for the activities of the next round dimer. This has been confirmed by Sarbeng et al. [108] by
[91]. It is very crucial to quote that a very small proportion carrying out four mutations on five residues on the interface
of protein substrates reach their native conformations fol- of the dimer. These mutations showed improper dimer folding
lowing an initial round of the GroEL reaction cycle. and decrease in the chaperone activity thereby signifying the
Accordingly, the molecules that have previously dissociated formation of a dimer in an ATP-dependent fashion. The
from GroEL in their non-native conformations go through reaction of DnaK begins by the recognition of a stretch of
another round of cycle to attain proper folding [92]. (~7) hydrophobic amino acids on the peptide substrate by
Moreover, the released non-native substrate proteins are SBDβ [109, 110]. This brings a conformational change in the
found to be the same or very similar to the ones bound SBDβ together with the SBDα, the affinity for the peptide
previously [93]. If the non-native proteins released by the substrate is regulated using ATP [111]. Two important con-
GroEL reaction cycle do not undergo another cycle, they are formations of DnaK, with respect to ATP attachment, favor its
recognized by some other cellular chaperonins or proteases mechanism of action. When ATP is bound to the NBD, it is
that decide the fate of the protein accordingly. known as the open state and the chaperone has a low affinity
for its substrate. The closed state, on the other hand is char-
DnaK acterized by ATP hydrolysis which enhances the affinity of
the SBD for the substrate [31, 112]. This shuffling between
DnaK in E. coli is the homolog of the eukaryotic Hsp70 the two conformations is brought about by the interaction
chaperone which functions in an ATP-dependent manner with the co-chaperones (DnaJ, TF and GrpE), as mentioned
[94]. The Hsp70 chaperone system is highly conserved earlier. DnaJ is said to interact with DnaK via its J-domain to
throughout the species i.e., from bacteria to humans [95]. induce the closed conformation to stabilize the substrate
This class of chaperones aids in protein remodeling and binding and GrpE binds after DnaJ dissociates from DnaK
activation reactions such as transportation, disaggregation catalyzing the dissociation of ADP by its hydrolysis
and folding [31, 96, 97]. DnaK is abundantly found in the [108, 113–115]. The substrate is finally released for refolding
E. coli cytosol and functions with the assistance of co- when ATP attaches to DnaK again, following GrpE dis-
chaperones such as DnaJ (a homolog of eukaryotic Hsp40 sociation and the cycle continues (Fig. 4c).
chaperone), TF (trigger factor) and a nucleotide exchange
factor, GrpE [98, 99]. E. coli essentially does not need ClpB
DnaK at intermediate temperatures for its growth, however
those that lack DnaK cannot withstand higher temperatures Caseinolytic peptidase B (ClpB) is a bacterial chaperone
and eventually die [100]. In several studies, DnaK has that belongs to the ATPase protein superfamily, AAA+
served as a great model for studying the structure and (ATPases Associated with diverse cellular Activities) and it
mechanism of Hsp70 chaperones. is a part of the eukaryotic Hsp100 chaperone group [116].
DnaK (Hsp70) is composed of two domains: NBD Hsp100 chaperones are present in many species such as
(nucleotide binding domain at the N-terminus) and SBD plants, fungi, bacteria, and protozoa [117]. ClpB is also
(substrate binding domain at the C-terminus), weighing ~45 called a disaggregase because it unravels proteins via its
kDa and 25 kDa, respectively. The SBD is further com- central pore by ATP hydrolysis [118, 119]. Under stress
posed of two subdomains: SBDα (α helical domain) and conditions (particularly high temperature), ClpB protects E.
SBDβ (β sandwich domain). The former being the lid and coli from protein aggregation and inactivation [120]. ClpB
the latter being the site for substrate attachment [31, 101– functions in cooperation with the DnaK/J system [121].
103] (Fig. 4a, b). Two lobes, I and II, each with IA, IB and ClpB also helps in the virulence of certain bacteria such as
IIA, IIB subdomains respectively, assemble to form the Francisella tularensis that causes tularemia, as reported by
NBD which contains an MgATP/MgADP binding site in a Alam et al. [122]. F. tularensis contains a T6SS (type VI
deep cleft [104]. The NBD is also known as the ATPase secretion system) which is linked to its pathogenicity. ClpB
domain. Both the NBD and SBD are linked by an inter- helps in the assembling and disassembling of T6SS.
domain linker consisting of a highly conserved sequence ClpB is a hexameric ring composed of three main
[105, 106]. This linker allows both the domains to function domains in every protomer: an NTD (N-terminal domain)
in an allosteric fashion. Just like any of the other proteins, and two nucleotide-binding domains (NBDs) AAA+
chaperones too have specific substrates which they help to domains (D1 and D2) known as Walker-type NBDs
gain their native conformations. DnaK does not bind to [123, 124] (Fig. 5a, b). The NTD is connected to D1 by a
native proteins, rather only unfolded, misfolded or partially linker that contains 17 residues which does not have a
folded proteins with exposed hydrophobic residues on the defined structure [125]. Both AAA+ domains contain
surface [107]. Walker A and Walker B motifs, respectively [120].
Fig. 4 Structure of DnaK.

a DnaK closed conformation
showing the domains and
subdomains. The cavity in the
NBD (nucleotide-binding
domain) is the site for ATP
attachment. (PDB ID: 2KHO
[167]). b DnaK open
conformation with ATP attached
at the NBD. The open lid allows
the SBDβ (substrate binding
domain) to bind to the incoming
substrate. (PDB ID: 4B9Q
[168]). c Schematic diagram of
the DnaK mechanism of action:
DnaJ and GrpE aid in the ATP-
driven unfolding of the
misfolded protein. The unfolded
protein is released to be refolded
or transferred to other chaperone
systems
D1 contains an M-domain (middle domain) which is a long, the aggregated proteins. Since ClpB is least likely to func-
coiled coil structure composed of two short coiled coils that tion without DnaK, Johnston et al. [131] determined the
run in opposite directions [126]. The M-domain is important innate substrate preference of ClpB without the presence of
for DnaK binding [126, 127]. Protein aggregates bind to the DnaK/J chaperone system. The AAA+ D1 domain deter-
NBD and the main body of the hexameric ring is sur- mines the substrate specificity which could be due to the
rounded by AAA+ D1 [116, 125, 128]. ATP hydrolysis and highly conserved pore loops of the domain or due to resi-
binding takes place at the NBD [129]. Substrate binding and dues that are transiently exposed during the conformational
interaction takes place at the NTD which is a globular changes by ATP association and disassociation. The con-
domain existing in helical conformation [120, 127]. cluding remarks suggest that different substrate preferences
Li et al. [123] investigated the mechanism behind the are exhibited by ClpB in the absence of DnaK.
binding of ClpB to various unstructured polypeptides. The As mentioned earlier, ClpB carries out the process of
first interaction was investigated between αS1casein and protein disaggregation with the help of DnaK/J system.
ClpB by modifying it to αS1casein-177 (177 amino acids) To understand the underlying mechanism behind the
which contains a single cysteine residue at the N-terminus ClpB–DnaK interaction, Durie et al. [121] conducted a
for it to be able to attach to a labeling substance, fluor- study on how DnaK induces changes in ClpB which
escein-5’-maleimide. The observations indicated that ClpB determines how ClpB interacts with its substrates. It was
existed in the form of monomers and hexamers and various found that the dissociation between the substrate and ClpB
oligomeric states are capable of binding to the substrate. is induced by DnaK, thereby serving as a peptide release
According to Ranaweera et al. [130], ClpB does not factor. Upon DnaK binding, a conformational change is
recognize the aggregated proteins based on local sequence induced in ClpB which makes it shift to its low-peptide
motifs, rather it considers the general surface properties of affinity state. To activate ClpB, two stimuli are needed:
Fig. 5 Structure and mechanism of action of ClpB complex. a ClpB rings of six NTDs (blue), NBD1-MD (cyan) and NBD2 (orange).
protomer structure: The ClpB protomer structure consists of NTD (N- c Schematic diagram of ClpB mechanism of action: ClpB is a hex-
terminal domain; blue), NBD1 and NBD2 (nucleotide binding domain americ ring-shaped chaperone that disentangles aggregated peptides
1 & 2; light blue & orange) and an M Domain (motif domain; cyan) with the help of the DnaK/DnaJ/GrpE chaperone system to yield
(PDB ID: 4D2X, Chain A [169]). b ClpB complex 3D structure: The unfolded peptides. These unfolded peptides can then either refold
protomers assemble to form a hexameric complex containing three properly on their own or can be directed to other chaperone systems
attachment of DnaK and binding of the substrate This, however, has been a great challenge for researchers.
[126, 127, 132, 133]. DnaK recruits the aggregated protein Over-production of recombinant proteins in E. coli has been
that binds to the M-domain of ClpB which allows ClpB to observed to form IBs. The production of IBs can be reduced
actively thread the aggregated protein through its central by molecular chaperones which ameliorates the soluble
pore in an ATP-driven fashion [126, 134]. The dis- expression of recombinant proteins. In this section, the role
aggregation process is carried out by extracting out single of trigger factor, GroEL, DnaK and ClpB in the soluble
polypeptide chains entangled in the aggregate and forcefully expression of recombinant proteins has been discussed.
unfolding those when they pass through the central pore
[119]. The individual polypeptides are gradually released Role of Trigger Factor
from the chaperone so they can obtain their native
conformation. As trigger factor (TF) possesses both PPIase and chaperone
activity, it has been co-expressed with various recombinant
proteins to enhance their soluble expression. TF has helped
Chaperone Assisted Recombinant Protein to attain the soluble yield of multiple recombinant proteins,
Expression either alone or in combination with other chaperones
(Table 2). For instance, a viral protein, CPV-VP2 (canine
Recombinant proteins are required in a large amount parvovirus virus protein 2), had been expressed in soluble
with maximum purity and solubility for structural studies. active form when co-expressed with trigger factor [135].
Table 2 Enhanced soluble expression of recombinant proteins with trigger factor co-expression
Recombinant protein Optimized conditions Co-chaperones Expression enhancement Results References
Spike protein of PEDV Inducer: 0.1 mM IPTG for DnaK/DnaJ/GrpE and 10 µg of purified protein COE and SP1 were When the proteins were co-expressed with TF, [139]
24 h at 15 °C GroEL/ES obtained they presented soluble expression as compared to
Tag: GST tag DnaK/DnaJ/GrpE and GroEL/ES
pH: 7.0–8.0
Der protein 2 Inducer: 0.3 mM IPTG for – 5 mg of pure Der p 2 obtained N-terminal histidine and trigger factor tagged [142]
20 h at 16 °C protein showed soluble expression
Tag: 6x His-tag
pH: 7.4
CPV protein VP2 Inducer: 0.1 mM IPTG for – At lower dosage of antigen 10 µg, Co-expression of CPV VP2 protein with trigger [135]
24 h at 25 °C antibodies can bind efficiently with antigen factor resulted in the active protein with
pH: 8.0 CPV VP2 protein. Thus, activating immune immunogenicity which can be used to produce
response CPV VLPs (virus like particles) with high yield
and purity
Xylose dehydrogenase and Inducer: 0.5 mM IPTG for GroEL/ES and DnaK/ 1.01 g/L 1, 2, 4-Butanetriol production was 1, 2, 4-Butanetrol titer was improved from 0.42 – [136]
benzoylformate 2 h at 37 °C L-arabinose DnaJ/GrpE obtained through trigger factor co- 0.56 g/L from xylose which was improved further
decarboxylase 20 mg/ml. expression at pH 7 at pH 7 under trigger factor co-expression system.
pH: 7.0 BT titer was reduced remarkably when expressed
with GroEL/ES and DnaK/DnaJ/GrpE
FMDV protein VP1 Inducer: 0.1 mM & – 1.5 g/l, 1.8 g/l, 0.8 g/l & 1.2 g/l yield of All of these proteins after vaccine booster had [138]
0.2 mM for 24 h at 18 °C purified soluble rF5V, rC4V, rV5F & rV4C induced 10 μg of antibodies against VP1 protein in
& 16 h at 25 °C were obtained guinea pigs
Tag: Calreticulin
pH: 9.0
CRM197 (Diphtheria toxin Inducer: 0.4 mM IPTG for GroEL/ES DnaK/ 150.69 ± 8.95 μg/ml of fCRM197 was Trigger factor co-expression successfully resulted [137]
mutant) 12 h at 25 °C DnaJ/GrpE obtained in high level production of pure soluble fCRM197
Tag: Trx, S and His tag
pH: 8.0
PEDV Spike protein Inducer: 0.3 mM IPTG for Flagellin-SO fusion TF improved 99% solubility of the fusion Truncated SO protein showed 66% solubility [140]
5 h at 25 °C protein protein while flagellin fused SO proteins were entirely
Tag: 6x His-tag soluble in the presence of trigger factor
pH: 12.5
Human Alpha folate Inducer: 0.4 mM IPTG for – 0.93 mg of soluble protein obtained Trigger factor fused tag was used with human FRα [143]
receptors 5 h at 37 °C proteins which remarkably enhances the soluble
Tag: Ni NTA tag yield of the protein. It was reported in previous
pH: 7.2 studies as inclusion bodies.
Gam1 Inducer: 0.1 mM IPTG for 6x His-TF-Gam1 and >90% pure soluble protein obtained Higher pH, reduction with DTT and cooling [141]
14 h at 16 °C TF-Gam1–6x His temperature before induction increased pure Gam1
Tag: His tag production. 6x His-tag at C-terminal of Gam1
pH: 7.7 protein gave better full-length purification results
Likewise, trigger factor also increased the activity of xylose along with a few solubility enhancing proteins including
dehydrogenase (Xdh) and benzoylformate decarboxylase maltose binding protein (MBP), glutathione S-transferase
(Xdh) which are important enzymes in the artificially (GST), thioredoxin (TRX) and trigger factor was used.
designed 1, 2, 4-butanetriol (BT) pathway. BT is a valuable Trigger factor fusion protein had remarkably enhanced the
chemical in the military industry and is artificially synthe- soluble expression of TonB protein while exhibiting a slight
sized in bacteria due to various hazards that arise with its improvement in scytovirin soluble expression as compared
chemical synthesis [136]. In the same manner, trigger factor to MBP, TRX and GST [144].
proved to be effective in the formulation of conjugated
vaccine carrier proteins such as CRM197 [137]. Role of GroEL
Over-expression of trigger factor also showed promising
results in the development of a vaccine against foot and GroEL/ES overproduction has proved to be a highly pro-
mouth disease virus by assisting in the correct folding of ductive method in E. coli system to counter the problems
immunogenic candidates (VP1). VP1 protein was fused that arise with protein folding. The GroEL/ES chaperone
with a truncated calreticulin (CRT) (Ca+ binding protein in system has been widely used to enhance the soluble
the endoplasmic reticulum (ER) lumen) at the N or C ter- expression of many recombinant proteins that are produced
minus followed by co-expression with trigger factor which in excess amount and form insoluble aggregates. The use of
resulted in high solubility of the fusion protein [138]. this chaperonin system facilitates successful expression of
Similarly, a flagellin protein fusion tag with a truncated various proteins in soluble proportions. These proteins
spike protein (SO) of porcine epidemic diarrhea virus include the human papillomavirus (HPV) L1 [145], N-acyl-
(PEDV) was also used to evaluate the ability of flagellin D-glucosamine 2-epimerase [146], human L-ferritin chain
fusion protein in enhancing the solubility and immuno- [143], 5-aminolevulinic acid (ALA) [147], TNF-related
genicity of SO. The co-expressed fusion protein with trigger apoptosis-inducing ligand (TRAIL) [148] and human anti-
factor exhibited up to 99% soluble expression along with EGFR ScFv antibody [149]. Several recombinant proteins
humoral immune response against SO [139]. that have shown an increment in their functional yield when
Trigger factor has also been used with various other co-expressed with GroEL/ES in the E. coli system have
chaperone systems such as GroEL/GroES and DnaK/DnaJ/ been listed in Table 3. For example, glyceraldehyde-3-
GrpE to obtain the soluble expression of GST-tagged fusion phosphate dehydrogenase is an important enzyme of the
proteins: rGST-COE (core neutralizing epitope) and rGST- glycolysis which was successfully obtained with ~5-fold
S1D (domain 1 of PEDV spike protein) [140]. Compared to increase in specific activity after GroEL/ES co-expression
all the chaperones, the highest soluble production of the [150]. Consistently, another strategy to obtain high yield
recombinant proteins was achieved by trigger factor. and low-cost production of ALA by hemA codon optimized
In several studies, trigger factor has been used as a fusion expression in the presence of the GroEL/ES complex has
protein tag to enhance the solubility of recombinant pro- been employed. This method has enhanced the ALA pro-
teins. Gam1 is an avian adenoviral replication protein which duction in the presence of GroEL/ES complex and TrxA
inhibits SUMOlyation of cellular proteins. Although Gam1 (thioredoxin) tag [147]. The HPV capsid protein L1 has
has been characterized in many studies but due to lack of its been of great importance in immunological studies as it can
structural details, it is difficult to investigate the properties be used as a potent vaccine candidate. L1 mostly forms IBs
of Gam1. Approximately 90% soluble expression of Gam1 when expressed in E. coli. This can be resolved by co-
was obtained by expressing it with trigger factor as a fusion expression with the GroEL/ES chaperone system to obtain
protein partner under cold shock promoter and low tem- ~3-fold increment in the soluble expression of L1 [145].
perature induction [141]. In the same way, trigger factor The co-expression of the GroEL/ES complex has also been
was fused with N-6x His tagged Der p 2 that is a clinically successful in recovering the native structure of TRAIL
relevant allergen present in Dermatophagoides pter- which is a potential tumor-targeting drug that induces tumor
onyssinus. Der p 2 was obtained in soluble, immunologi- death by stimulating apoptosis [148].
cally active form with high purity [142]. Alpha fetoprotein Various research studies have shown greater successful
receptors have been extensively studied due to their role in results when the GroEL/ES complex was used in combi-
cancer development and diagnosis. FRα was also fused with nation or in comparison with other molecular chaperones
trigger factor which resulted in the higher expression of the such as Hsp70 (DnaK) or trigger factor. Ferritins are used as
recombinant fusion protein and confirmed its presence in therapeutic carrier nano-cage delivery systems in medical
the soluble cell lysate fraction [143]. Moreover, in another imaging and therapy. However, when ferritins are synthe-
study, a unique set of vectors was designed to obtain an sized in heterologous expression systems, they become
optimized soluble expression of TonB CTD and scytovirin prone to the formation of IBs. His tagged L-ferritin [151]
viral protein. A yeast SUMO tag fusion protein system and H-ferritin [152] were co-expressed in E. coli with
Table 3 Enhanced soluble expression of recombinant proteins with GroEL co-expression
Recombinant Protein Optimized Conditions Co-Factors Expression Enhancement Results References
Tumor necrosis factor-related Inducer: 30 °C induction till GroES Only few recoverable polypeptides After recovery of in vivo TRAIL inclusion bodies by [148]
apoptosis inducing ligand 0.6 OD600, then further for found as inclusion bodies GroEL/ES, IBs retain a lower level of recoverable
4 h at 42 °C polypeptides and anti-tumor activity. These results
pH: 8.0 showed that GroEL/ES proved to be helpful in
in vivo refolding of IBs
hemA Inducer: 0.1 mM IPTG GroES and ATP 1.21–3.67 g/l improvement in ALA Following a two-stage induction strategy and co- [147]
induction for 24 h at 30 °C production expression with GroEL, a significant increment was
Tag: TrxA observed in the production of ALA
pH: 4.6
HPV capsid protein L1 Inducer: 0.2 mM IPTG & GroES, 0–8 mM ATP ~3-fold increase in expression of Results confirmed by circular dichroism, indicating [145]
1 mg/ml induction for 18 h &0–20 mM MgCl2 GST-fused L1 protein with GroEL/ES that L1-p possesses properties (structural
at 28 °C conformation and biological activity) of native L1
Tag: GST
pH: 8.0
Glucosamine 2-epimerase Inducer: 0.1 & 1 mM IPTG GroES and 264-fold increase in activity Higher soluble expression of protein was obtained at [146]
Induction for 6 & 8 h at 100 µM ATP higher temperature (37 °C) compared to initial
20 °C conditions (20 °C) when co-expressed with GroEL/
Tag: 6x His-tag ES chaperonin
pH: 6.5–7.5
Sarcosine oxidase Inducer: 0.5 mmol/L IPTG GroES ~10-fold increase in soluble Improved expression of codon-optimized SOX [156]
at 37 °C for 8 h expression
pH: 8.0
Human Ferritin L-Chain Inducer: 37 °C for 5 h, GroES not ATP 3-fold increase in the presence of Co-expression of H-chain ferritin with chaperones is [151]
1 mM IPTG chaperonin essential for soluble expression
Tag: 6X His-Tag
Glyceraldehyde–3-Phosphate Inducer: 5 mM IPTG GroES not ATP 4-fold increase in soluble Enhanced enzyme activity after expression with [150]
dehydrogenase Induction at 25 °C concentration GroEL/ES
Tag: 6x His-tag
pH: 7.4
C-X-C Chemokine Inducer: Cell-free GroES ~30-fold increase in activity and ~1.3- Soluble expression of biologically active CXCR4 [174]
GPCR type 4 expression, no induction fold increase in yield
Tag: 6X His-Tag
pH: 7.2
Human H-chain Ferritin Inducer: 1 mM IPTG GroES >3-fold solubility of human H-chain rHF efficiently folded in the GroEL nanocage [152]
Induction at 20 °C for 15 h ferritin
Tag: 6x His-tag
pH: 7.4
D-hydantoinase Inducer: 0.1 mM IPTG GroES 78.3% isolation yield Increase specific activity of GroEL-co-expressed [154]
Induction at 25 °C for 6 h hydantoinase
Tag: 6x His-tag
pH: 9.0
GroEL/ES complex and trigger factor to facilitate their

References native folding. A six-chaperone co-expression system was
[153]
High amount of soluble CRM197 by co-expression [155]

also employed to attain high soluble and low-cost produc-
tion of polyunsaturated fatty-acid isomerase (PAI) which is
involved in conjugated linoleic acid (CLA) synthesis.
Combined expression of GroEL/ES with DnaK/J/GrpE
increased the activity by 11.6%. Although GroEL alone had
remarkably shown improvement (57.8%) in PAI activity,
Improved PAI solubility and activity
the activity further increased to 80% after optimization of

conditions [153].
with GroEL and trigger factor
D-hydantoinase belongs to the dihydropyridiminase

class of enzymes which catalyze the biosynthesis of D-
amino acids. Five different plasmids with five sets of
recombinant chaperones were used to obtain the soluble
expression of D-PfHYD. Under the expression of GroEL/
ES, DPfHYD was expressed in soluble form with highest
Results
specific activity as compared to other chaperone combi-

nations [154]. A pure and nontoxic Diphtheria toxin
mutant, CRM197 was also successfully expressed by co-
improved the solubility from 29% to
expressing with different molecular chaperones. The

highest soluble amount of the recombinant mutant was
111.24 ± 10.40 μg/ml soluble
Co-expression of GroEL/ES
obtained using GroEL/ES and trigger factor [155]. N-acyl-

97% and activity by 57.8%
Expression Enhancement
D-glucoseamine-2-epimerase is required for large scale

CRM197EK obtained
synthesis of N-acetylneuraminic acid but due to the for-

mation of insoluble aggregates, low yield is obtained. The
use of a combination of different prokaryotic chaperones
with the GroEL/ES complex exhibited approximately a
264-fold increment in the epimerase activity [146]. In
another study, GroEL co-expression strategy was imple-
mented proficiently to obtain a tenfold increase in the
solubility of sarcosine oxidase [156].
Role of DnaK
Co-Factors
GroES
GroES
Technology for producing recombinant proteins is rapidly

expanding with time and extensive work is being carried out
Tag: 6x His-tag and Trx tag
Induction at 20 °C for 24 h
for protein production. High-scale protein production often

Inducer: 0.1 mM IPTG &
results in the formation of IBs causing loss of protein

Inducer: 0.1 mM IPTG
1.5 mg/ml induction at
Optimized Conditions
structure and function. This can be rectified using molecular

chaperones. DnaK has been used in various studies to bring
20 °C for 20 h
about folding of proteins into their native forms (Table 4). It

has been observed that the presence of DnaK showed sig-
pH: 7.4
pH: 8.0
nificant increase in the refolding of denatured carbonic

anhydrase followed by an 80% increase in enzyme activity
[157]. Moreover, E. coli strains containing the recombinant
DnaK showed a higher survival rate in salt conditions
Polyunsaturated fatty acid
compared to the ones that had not been transformed.

Recombinant Protein
The co-expression of DnaK/J system and other chaper-

Table 3 (continued)
one systems with recombinant co-type nitrile hydratase

isomerase (PAI)
(NHase) [158], manganese peroxidase [159] and anti-TNF-

α Fab antibody [160] substantially enhanced the activity
CRM197
and solubility of the respective proteins. The highest

increase in the solubility of anti-TNF-α Fab antibody was
due to the DnaK/J chaperone system. Likewise, the co-

References expression of DnaK and its co-chaperones DnaJ and GrpE
[157]
[158]
[160]
[161]
[163]
[159]
[164]
with GM-CSF (granulocyte-macrophage colony-stimulating
factor) [161], rhFGF21 [162] and humanized anti-CD20
scFv (single chain variable fragments) [163] greatly
36% soluble fraction of anti-TNF-α Fab antibody in

The resultant refolding assay showed a considerable
enhanced protein soluble expression.

improvement in the refolding rate of heat denatured
50% increase in expression of soluble anti-CD20
periplasm and 47% cytoplasmic soluble fraction

Similar to the aforementioned study regarding anti-
TNF-α Fab antibody, another study also reported the effect
Improved expression of soluble GM-CSF

of co-expression of DnaK/J system and DsbC chaperone
23.2% soluble Fab antibody produced
Increase in MnP soluble expression

on the expression and translocation of anti-TNF-α Fab
antibody in the E. coli periplasm [164]. The DnaK/J sys-
NHase was actively expressed
tem co-expression exhibited a 2.5-fold increase in the anti-

TNF-α Fab antibody translocation.
recombinant protein
carbonic anhydrase
Role of ClpB
Results
Due to the availability of different chaperone systems,

ClpB so far has not been particularly used in recombinant
technology to enhance protein solubility. ClpB mainly
2.5-fold increase in the number of E. coli
80% increase in heat denatured carbonic
antibody translocation to the periplasm

52-fold higher activity after expression
requires DnaK to carry out its function. When used in

2.5-fold increase in anti-TNF-α Fab
with co-chaperones DnaJ and GrpE
Sevenfold increase in Fab antibody
325 μg/mL soluble anti-CD20 scFv

Up to threefold increase in soluble
combination with the DnaK/J and GroEL/ES chaperone

systems, ClpB is shown to increase the yield of soluble
Expression Enhancement
recombinant proteins [165].

colonies with DnaK
anhydrase activity
Conclusion
expressed
proteins
activity
The production of recombinant proteins in E. coli possesses

–
a lot of challenges when carried out in high amounts and the

Table 4 Enhanced soluble expression of recombinant proteins with DnaK co-expression
DnaJ, GrpE
Inducer: 0.1 mM IPTG for 6 h at 25 °C DnaJ, GrpE
DnaJ, GrpE
DnaJ, GrpE
Inducer: 1 mM IPTG for 2 h at 30 °C, 5 h DnaJ, GrpE
Inducer: 0.1 mM – 1 mM IPTG for 8 and DnaJ, GrpE
most repeatedly occurring is the formation of IBs. As E. coli

Co-Factors
is mainly used as the host for recombinant protein produc-

tion; often its own cellular proteins hinder the production of
Inducer: 0.1 mM IPTG for 2, 4, and 6 h at –
large quantities of recombinant proteins. The most advanced

at 25 °C & overnight at 20 °C and 16 °C
Inducer: 0.5 mM IPTG for 4 h at 37 °C
and promising technique to combat this issue is the use of

Inducer: 1 mM IPTG for 4 h at 30 °C
molecular chaperones as co-expressed companions of

Inducer: 100 µmol IPTG at 18 °C
recombinant proteins. E. coli encodes different chaperones,

out of which, some are involved in folding and the rest in
Optimized Conditions
protein disaggregation. Molecular chaperones have been

reported to be effective in the soluble expression of various
30 °C and 37 °C
Tag: 6x His-tag
Tag: 6x His-tag
Tag: 6x His-tag
Tag: 6x His-tag
16 h at 25 °C
heterologous proteins. Trigger factor, GroEL and DnaK

pH: 7.75
exhibited a significant increase in the expression of several

pH: 8.0
recombinant proteins, either alone or in combination with

each other. ClpB has not yet been used alone to solubilize
Humanized anti-CD20 scFv (single chain
recombinant protein expression as it requires the assistance

of DnaK to function. Although, there is no definite panacea
Co-type nitrile hydratase (NHase)
Granulocyte-macrophage colony-
to solve the difficulties that arise during protein folding,

Manganese peroxidase (MnP)
stimulating factor (GM-CSF)
molecular biologists are increasingly turning their research

Anti-TNF-α Fab antibody
Anti-TNF-α Fab antibody
towards chaperone co-expression systems as their first-line

Recombinant Protein
Carbonic anhydrase
variable fragments)
strategy to overdraw protein folding challenges.
Compliance with Ethical Standards
Conflict of Interest The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to Microbial Cell Factories, 4(1), 1 https://doi.org/10.1186/1475-
jurisdictional claims in published maps and institutional affiliations. 2859-4-1.
17. Nguyen, H. D., Nguyen, Q. A., Ferreira, R. C., Ferreira, L. C. S.,
Tran, L. T., & Schumann, W. (2005). Construction of plasmid-
based expression vectors for Bacillus subtilis exhibiting full
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A Review: Molecular Chaperone-Mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins

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Cell Biochemistry and Biophysics

A Review: Molecular Chaperone-mediated Folding, Unfolding and

Received: 22 July 2020 / Accepted: 1 February 2021

Introduction handle genetic modiﬁcations due to convenient selection of

Fig. 4 Structure of DnaK.

GroEL/ES complex and trigger factor to facilitate their

High amount of soluble CRM197 by co-expression [155]

the activity further increased to 80% after optimization of

D-hydantoinase belongs to the dihydropyridiminase

speciﬁc activity as compared to other chaperone combi-

expressing with different molecular chaperones. The

obtained using GroEL/ES and trigger factor [155]. N-acyl-

D-glucoseamine-2-epimerase is required for large scale

synthesis of N-acetylneuraminic acid but due to the for-

Technology for producing recombinant proteins is rapidly

for protein production. High-scale protein production often

results in the formation of IBs causing loss of protein

structure and function. This can be rectiﬁed using molecular

about folding of proteins into their native forms (Table 4). It

niﬁcant increase in the refolding of denatured carbonic

compared to the ones that had not been transformed.

The co-expression of DnaK/J system and other chaper-

one systems with recombinant co-type nitrile hydratase

(NHase) [158], manganese peroxidase [159] and anti-TNF-

and solubility of the respective proteins. The highest

due to the DnaK/J chaperone system. Likewise, the co-

36% soluble fraction of anti-TNF-α Fab antibody in

enhanced protein soluble expression.

50% increase in expression of soluble anti-CD20

periplasm and 47% cytoplasmic soluble fraction

Improved expression of soluble GM-CSF

Increase in MnP soluble expression

tem co-expression exhibited a 2.5-fold increase in the anti-

Due to the availability of different chaperone systems,

antibody translocation to the periplasm

requires DnaK to carry out its function. When used in

Sevenfold increase in Fab antibody

325 μg/mL soluble anti-CD20 scFv

combination with the DnaK/J and GroEL/ES chaperone

recombinant proteins [165].

The production of recombinant proteins in E. coli possesses

a lot of challenges when carried out in high amounts and the

Inducer: 0.1 mM IPTG for 6 h at 25 °C DnaJ, GrpE

Inducer: 1 mM IPTG for 2 h at 30 °C, 5 h DnaJ, GrpE

Inducer: 0.1 mM – 1 mM IPTG for 8 and DnaJ, GrpE

most repeatedly occurring is the formation of IBs. As E. coli

is mainly used as the host for recombinant protein produc-

large quantities of recombinant proteins. The most advanced

and promising technique to combat this issue is the use of

molecular chaperones as co-expressed companions of

recombinant proteins. E. coli encodes different chaperones,

protein disaggregation. Molecular chaperones have been

heterologous proteins. Trigger factor, GroEL and DnaK

exhibited a signiﬁcant increase in the expression of several

recombinant proteins, either alone or in combination with

recombinant protein expression as it requires the assistance

to solve the difﬁculties that arise during protein folding,

molecular biologists are increasingly turning their research

Anti-TNF-α Fab antibody