Review
Production of disulﬁde-bonded proteins in Escherichia coli
Mehmet Berkmen ⇑
New England Biolabs, 240 County Road, Ipswich, MA 01938, USA
a r t i c l e i n f o
Article history:
Received 19 October 2011
and in revised form 24 October 2011
Available online 7 November 2011
Keywords:
Disulﬁde bond
Escherichia coli
Dsb
Origami
SHufﬂe
FA113
Oxidative protein folding
a b s t r a c t
Disulﬁde bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A
disulﬁde bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem
to the process of disulﬁde bond formation: mis-pairing of cysteines can cause misfolding, aggregation and
ultimately result in low yields during protein production. Recent developments in the understanding of
the mechanisms involved in the formation of disulﬁde bonds have allowed the research community to
engineer and develop methods to produce multi-disulﬁde-bonded proteins to high yields. This review
attempts to highlight the mechanisms responsible for disulﬁde bond formation in Escherichia coli, both
in its native periplasmic compartment in wild-type strains and in the genetically modiﬁed cytoplasm
of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles
involved in the formation of disulﬁde-bonded proteins with the hope of guiding the scientist in
choosing the optimum expression system.
Ó 2011 Elsevier Inc.
Contents
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
On the nature of disulfide bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Does my protein of interest have disulfide bonds? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Conservation and number of cysteines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Extra-cytoplasmic location of the protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Production of disulfide-bonded proteins in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Periplasmic production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Oxidation: DsbAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Reduction/isomerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Alternative mechanisms of periplasmic disulfide bond formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Alternative oxidoreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Engineered oxidoreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Fusion partners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Cytoplasmic production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Alternative mechanisms of cytoplasmic disulfide bond formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Overview
The study of protein disulﬁde bond formation began half a century
ago with Anﬁnsen’s seminal work on the folding of bovine
pancreatic ribonuclease (RNaseA).1
Anﬁnsen and colleagues demonstrated
that a fully denatured RNaseA having all three of its
disulﬁde bonds reduced, could re-fold in vitro back to its active correctly
folded disulﬁde-bonded state, independent of any help from
1046-5928 Ó 2011 Elsevier Inc.
doi:10.1016/j.pep.2011.10.009
⇑ Fax: +1 978 921 1350.
E-mail address: berkmen@neb.com
1
Abbreviations used: ER, endoplasmic reticulum; Trx1, thioredoxin 1; ROS, reactive
oxygen species; Dsb, disulﬁde bond; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PhoA, oxidize the alkaline phosphatase; RNR, ribonucleotide reductase.
Protein Expression and Puriﬁcation 82 (2012) 240–251
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journal homepage: www.elsevier.com/locate/yprep
Open access under CC BY-NC-ND license.
Open access under CC BY-NC-ND license.
cytoplasmic constituents [1]. Their conclusion was that the information
present within a polypeptide was sufﬁcient for it to achieve
its ﬁnal correctly folded state [2]. This led to the erroneous conclusion
that disulﬁde bonds are formed spontaneously within a living
cell. For the next three decades, their work sealed the notion that
an electron acceptor such as oxygen was sufﬁcient for catalyzing
disulﬁde bonds in vivo. This dogma was overturned in 1991 with
the discovery of a periplasmic enzyme from Escherichia coli required
for catalyzing disulﬁde bonds [3]. This discovery led to a
slew of investigations using several model organisms, resulting in
today’s detailed and sophisticated understanding of the biochemical
and biophysical processes that govern the formation (oxidation),
breaking (reduction) and shufﬂing (isomerization) of
disulﬁde bonds within a polypeptide in vivo. Several excellent reviews
have summarized the latest ﬁndings both in prokaryotes
[4–6] and in eukaryotes [7–10].
Despite the advances in eukaryotic expression systems, E. coli
remains a popular host for recombinant protein production both
in research and in biotechnological industry. For example, of the
58 biopharmaceuticals products approved from 2006 to 2010, 17
are produced in E. coli, 32 are produced in mammalian cell lines
(mainly Chinese hamster ovary; CHO) and four are produced in
Saccharomyces cerevisiae, transgenic animals, or via direct synthesis
[11]. The production of proteins to high yields remains a process
of trial and error. Given an uncharacterized protein, researchers
have few predictive tools to choose the correct host, expression
system and expression conditions. The numerous possible posttranslational
modiﬁcations that proteins can undergo [12] increase
the chances of failure at expressing a soluble active protein.
Researchers usually resort to trying as many expression solutions
as possible within their allocated time and resources. E. coli is often
the ﬁrst host of choice for most researchers, as overproduction and
puriﬁcation of a recombinant protein from E. coli can be faster,
cheaper, and easier than from other organisms. Furthermore, a
myriad of tools have been developed for the expression of proteins
in E. coli and a large body of information on protein folding solutions
is available.
This article will focus on reviewing the current understanding
of the processes that regulate the formation of disulﬁde bonds
within the model prokaryotic host E. coli. Emphasis will be given
to the utilitarian application of this knowledge to furthering the
capacity to heterologously express multi-disulﬁde-bonded proteins
in E. coli.
On the nature of disulﬁde bonds
Disulﬁde bonds are formed by the oxidation of thiol groups (SH)
between two cysteine residues resulting in a covalent bond. Disulﬁde-bonded
proteins are much more abundant than previously
appreciated. Analysis of the human genome has predicted that
more than half of the predicted proteins that are destined for the
endoplasmic reticulum (ER) and beyond acquire disulﬁde bonds
[13]. In the context of protein folding, disulﬁde bonds can be reduced,
oxidized or isomerized. These processes require an oxidoreductase
to catalyze an electron transfer event, during which a
short-lived disulﬁde-bonded complex forms between the substrate
and the enzyme (Fig. 1). All such oxidoreductases contain a thioredoxin
fold with a CxxC active site motif, where x is any amino acid.
Many factors determine whether, in vivo, an oxidoreductase is an
oxidase or a reductase. These factors include the relative stabilities
of the enzyme’s reduced and oxidized states, the compartment in
which the enzyme resides, and to some extent the amino acids
within the enzyme’s active site. Reductases, such as thioredoxin,
are usually more stable when their active site cysteines are oxidized
compared to when they are reduced [14] and vice versa for
Fig. 1. Thiol-disulﬁde bond exchange. Schematic representation of electron transfer between a substrate protein (grey ball) and an oxidoreductase (black line) resulting in
oxidation, reduction or isomerization of disulﬁde bonds.
M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251 241
oxidizing enzymes such as DsbA [15]. For example, a cytoplasmic
reductase such as thioredoxin 1 (Trx1) can act as an oxidase when
expressed in the periplasm, where it is decoupled from the reductases
which maintain it in its reduced state [16]. Manipulating the
amino acids near the active site pocket [17] or within the CxxC motif
[18] can also have profound effects on the reducing vs. oxidizing
activity of an oxidoreductase [19].
Disulﬁde bonds have multiple biologically signiﬁcant functions.
For example, disulﬁde bonds can play structural roles important
for the folding and stability of a protein. Disulﬁde bonds can also
act as signals of the redox environment; they can be readily oxidized
or reduced, thereby changing the conformation of the protein
in response to the redox state of the environment. Finally, in the
context of the active site of an oxidoreductase, cysteines can donate
or accept electrons, catalyzing either the oxidation or reduction
of disulﬁde bonds in a substrate protein. It is therefore
important for the researcher to be aware of the types of disulﬁde
bonds that exist in proteins: structural, signaling and catalytic.
Structural disulﬁde bonds decrease the possible number of conformations
for a given protein, resulting in decreased entropy and
increased thermostability [20]. For some proteins, disulﬁde bonds
are critical for their folding as reducing their disulﬁde bonds can
result in denatured inactive protein [21]. For other proteins, disulﬁde
bonds are not essential for their folding; instead they increase
the proteins’ stability [22,23]. Recombinant expression of proteins
in E. coli sometimes results in the formation of aberrant disulﬁde
bonds. One labor-intensive solution to this problem is to mutate
the non-essential cysteines that form the aberrant disulﬁde bonds
to produce greater yields of correctly oxidized soluble protein [24].
A more attractive general solution to this problem would be to express
the protein in a mutant E. coli strain background engineered
to possess alternative mechanisms of oxidative folding.
Proteins residing outside the protective chaperone-rich environment
of the cytoplasm are especially susceptible to environmental
abuse and are therefore usually rich in disulﬁde bonds to
increase their stability. Extra-cytoplasmic and surface associated
proteins are common therapeutic targets, and consist exclusively
or partially of disulﬁde-bonded proteins [25]. Furthermore, more
stable versions of industrially relevant proteins have been engineered
by inserting cysteines at speciﬁc locations to allow disulﬁde
bonds to form [26,27]. In nature, hyperthermophilic organisms use
the stabilizing effects of disulﬁde bonds to increase the thermostability
of their proteome. Crenarchaeal organisms thrive at temperatures
75° C to 105° C and have proteomes with enriched cysteine
content, possibly due to the higher frequency of disulﬁde-bonded
proteins [28]. For example, the enzyme adenylosuccinate lyase
has three disulﬁde bonds in the hyperthermophilic crenarchaeal
Pyrobaculum aerophilum, but lacks disulﬁde bonds in a mesophilic
organism [29]. Similar observations have been made for proteins
from other thermophilic organisms [30–32], including the DNA
polymerase from Thermococus gorgonarius [33]. It should also be
noted that structural cysteines can also be involved in coordinating
metal ions such as iron [34], mercury [35], zinc [36] or binding
prosthetic groups such as hemes [37] or FAD [38].
Signaling disulﬁde bonds are responsible for conveying the redox
state of their environment via conformational changes incurred in
the protein, during the oxidation and reduction of its redox-sensitive
disulﬁde bond(s). Due to their reversible nature, signaling
disulﬁde bonds are sensitive to their redox environment, and thus
tend to be transient in nature. For example, the transcription factor
OxyR which responds to cellular hydrogen peroxide has one signaling
disulﬁde bond that senses reactive oxygen species (ROS) within
the cytoplasm. Upon exposure to ROS, a transient disulﬁde bond is
formed, activating OxyR to transcriptionally initiate the oxidative
stress response. Removal of the oxidative stress results in the
reduction of the disulﬁde bond, returning OxyR back to its quiescent
state [39]. Other bacterial examples of signaling disulﬁde
bonds are found in the ArcAB two-component system which senses
and responds to changes in respiratory growth conditions of the
cell [40] and the cytoplasmic anti-sigma factor RsrA, responsible
for activating the cytoplasmic oxidative stress response [41].
Catalytic disulﬁde bonds are involved in redox reactions and are
found in all oxidoreductases. Catalytic disulﬁde bonds are usually
found within the classic CxxC active site motif. Oxidoreductases
that are involved in the oxidation of cysteines must have their active
site cysteines maintained in their oxidized, disulﬁde-bonded
state; one example of such an enzyme is the periplasmic DsbA
[42]. Conversely, oxidoreductases that are involved in the reduction
of disulﬁde bonds must have their active site cysteines maintained
in their reduced, thiolate states; examples of such enzymes
include the cytoplasmic oxidoreductases thioredoxin and glutaredoxin
[43]. The correct redox state of an oxidoreductase is usually
maintained by a partner protein (see Section c for more details).
It is sometimes difﬁcult to predict whether an oxidoreductase is
a dedicated oxidase or a reductase. One clue for the role of an oxidoreductase
comes from the enzyme’s cellular location. Oxidoreductases
involved in the formation of disulﬁde bonds are usually
found in extra-cytoplasmic compartments, such as the periplasm
in gram negative prokaryotes [6] and the endoplasmic reticulum
in eukaryotes [9]. Recent ﬁndings have also elucidated speciﬁc oxidases
in the inter-membrane space of mitochondria [44,45] and
the thylakoids of chloroplasts [46]. Furthermore, the redox environment
in which the oxidoreductase is expressed will affect the
redox state of the catalytic disulﬁde bond. For example, the cytoplasmic
reductase TrxA whose active site cysteines are maintained
in their reduced state will convert partially to an oxidase, when expressed
in the periplasm in vivo [47]. Additionally, amino acids
within its active site [19,48] and those of other oxidoreductases
that it interacts with, also play a signiﬁcant role in maintaining
the redox state of TrxA’s catalytic disulﬁde bonds [49,50].
Does my protein of interest have disulﬁde bonds?
When expressing a protein with unknown folding requirements,
a researcher is challenged to ﬁnd the appropriate expression
host and conditions, especially if the protein requires posttranslational
modiﬁcations such as disulﬁde bonds. It is therefore
critical to know whether a given protein contains disulﬁde bonds.
Web based servers [51,52] and algorithms [53,54] claim they can
predict the presence of disulﬁde bonds with up to 87% conﬁdence
[55]. However, such prediction programs are not yet widely used.
As an alternative simple method, I suggest that if a protein of interest
contains an even number of conserved cysteines and is predicted
to be extracytoplasmic, then the protein likely contains
disulﬁde bonds. The reasoning behind this is outlined below.
Conservation and number of cysteines
It was observed early on [56,57] that cysteine is one of the most
reactive amino acids and therefore one of the least abundant amino
acids within proteins [58]. In fact in Drosophila, UGU coding for
cysteine is the most infrequently used codon [59]. This feature of
cysteines is advantageous when used as a predictor of biological
function as their presence is less likely due to random mutation,
but more so a representation of their biological roles. Highly conserved
cysteines are often indicative of structural disulﬁde bonds,
especially if the conservation is within proteins from diverse
organisms. Since a disulﬁde bond requires the pairing of two cysteines,
generally proteins expressed in oxidative environments
tend to have even numbered cysteines. This correlation was used
to predict the presence of disulﬁde-bonded proteins in the cyto-
242 M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251
plasm of various thermophilic organisms [28,60] and the periplasm
of prokaryotes [61].
Extra-cytoplasmic location of the protein
In prokaryotes and eukaryotes, disulﬁde bond formation is compartmentalized
to non-cytoplasmic locations such as the periplasm
or ER, respectively. Proteins that require disulﬁde bonds for their
folding are secreted to these compartments and therefore harbor
an N-terminal signal peptide. There are several web based algorithms
that can predict the presence of a signal peptide to high levels
of conﬁdence [62,63]. However, not all periplasms are
oxidative. For example, the periplasm of the anaerobic Bacteroides
is predicted to be a reducing compartment [49,64]. In summary, a
secreted protein with conserved even-numbered cysteines is predicted
to contain disulﬁde bonds.
Production of disulﬁde-bonded proteins in E. coli
Periplasmic production
The cytoplasm of wild-type E. coli is not permissive to the formation
of disulﬁde-bonded proteins due to the presence of numerous
reductases and reducing agents such as glutathione. A protein
that requires disulﬁde bonds for its folding must be secreted to the
periplasm. There, a set of cell envelope proteins (named Dsb for
disulﬁde bond) catalyze the formation and correction of disulﬁde
bonds. The Dsb system has been studied in great detail for the last
two decades and several comprehensive reviews have been written
on this subject [6,65–67]. This section will give a brief summary
of these ﬁndings.
Oxidation: DsbAB
In E. coli, the protein machinery that catalyzes the formation of
disulﬁde bonds is localized in the periplasm (Fig. 2). DsbA is a
21 kDa monomeric protein whose in vivo and in vitro properties
have been extensively characterized. DsbA contains the classic thioredoxin
fold [68] and a CxxC active site motif. DsbA is a potent
oxidase [69], catalyzing the formation of disulﬁde bonds as a substrate
protein enters the periplasm [17]. After donating its disulﬁde
bond to the substrate, DsbA becomes reduced and must be re-oxidized
back to its active oxidized state by the inner membrane protein
DsbB [42]. DsbB, in turn, donates the electrons to either
ubiquinone during aerobic growth or to menaquinione during
anaerobic growth [70].
DsbA is an indiscriminately powerful oxidase and will catalyze
the formation of disulﬁde bonds, even in proteins that naturally do
not have disulﬁde bonds. For example, when the normally reduced
beta-lactamase CcrA from Bacteroides fragilis is expressed in the
periplasm of E. coli, its cysteines are oxidized rendering the protein
Fig. 2. Periplasmic disulﬁde bond formation. r A substrate protein which requires disulﬁde bonds for its folding is exported to the periplasm in its reduced non-disulﬁdebonded
state (ProtRED), usually via the sec pathway. s DsbA forms a disulﬁde-bonded complex with its reduced substrate resulting in the formation of a disulﬁde bond in the
substrate protein (ProtOXI) and the reduction of DsbA’s active site cysteines. t DsbA is re-oxidized by the inner membrane protein DsbB, which donates the electrons it has
received from DsbA either to ubiquinone (UQ) in aerobic conditions or to menaquinione (MQ) in anaerobic conditions. u If the substrate protein is mis-oxidized (ProtMIS-OXI)
by DsbA, the misfolded protein is recognized by DsbC and is either isomerized to its native state or reduced, allowing DsbA to have another chance at correctly oxidizing the
protein. v DsbC is maintained in its active reduced state by the inner membrane protein DsbD. w DsbD receives its electrons from the cytoplasmic thioredoxin (TrxA) which
ultimately receives its electrons from the cytoplasmic pool of NADPH. Cysteines are represented as yellow balls in their reduced state or as sticks in their disulﬁde-bonded
state. Grey arrows show products obtained after each step, whereas red arrows represent electron ﬂow. (For interpretation of the references to colour in this ﬁgure legend, the
reader is referred to the web version of this article.)
M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251 243
inactive [71]. Similarly, when cytosolic proteins whose cysteines
are naturally maintained reduced are exported to the periplasm,
they too become oxidized by DsbA and are misfolded [3].
Reduction/isomerization
DsbCD. Proteins that are mis-oxidized, and are therefore misfolded,
may be isomerized back to their native disulﬁde-bonded
state by DsbC [72,73] (Fig 2). DsbC is a 23 kDa homodimeric protein
consisting of two domains, a thioredoxin and a dimerization
domain, linked by a short alpha-helical strand [74]. The thioredoxin
domain of DsbC contains four cysteines. The carboxyl-terminal
cysteine pair forms a structural disulﬁde bond that is crucial for
the stability of DsbC [75]. The amino-terminal cysteine pairs are
part of the active site CxxC motif and are maintained in their
reduced state by the inner membrane protein DsbD [76]. DsbD
receives its reducing potential from cytoplasmic thioredoxin,
which ultimately receives its electrons from the pool of NADPH
[77].
Why are some proteins correctly oxidized by DsbA while others
are not? Since DsbA rapidly and efﬁciently catalyzes disulﬁde bond
formation as an unfolded protein is translocated into the periplasm
[17], it has the tendency to oxidize cysteines in a consecutive manner
[17,78]. Proteins that require non-consecutive disulﬁde bonds
are thus often misfolded, and may be either degraded by periplasmic
proteases or isomerized back to their correct oxidized state by
DsbC. Table 1 shows a list of characterized proteins, their disulﬁde
bond connectivity patterns, and their dependency on DsbC for
correct folding. Proteins such as Hcp having eight cysteines (four
consecutive disulﬁde bonds) and therefore 105 possible disulﬁdebonded
states is correctly oxidized by DsbA [79], while MGSA with
four cysteines (two non-consecutive disulﬁde bonds) and only
three possible disulﬁde-bonded states, is not [80]. In general,
proteins with consecutive disulﬁde bonds do not require DsbC
while proteins with nonconsecutive disulﬁde bonds do. Although
it is likely that exceptions to this correlation might exist in nature,
this reviewer has yet to ﬁnd a documented exception.
Disulﬁde bond isomerization is crucial for the production of
nonconsecutive multi-disulﬁde-bonded proteins in the periplasm.
Over-expression of DsbC in the periplasm can result in substantial
improvement in yields of correctly folded proteins [81–86]. Our
current understanding of the in vivo mechanism of disulﬁde bond
isomerization remains limited. Two mechanistic questions about
DsbC remain elusive and are central to our understanding of disulﬁde
bond isomerization in vivo: How does DsbC discriminate between
correctly folded proteins and those that are mis-oxidized?
How does DsbC isomerize a mis-oxidized protein back to its native
state?
Clues to DsbC’s substrate recognition were eventually found
upon solving its crystal structure. Dimerization of the two monomers
of DsbC results in a ‘‘V’’ shaped protein with an uncharged
cleft 38 Å wide [74]. Another folding chaperone, trigger factor, with
a hydrophobic cleft of similar dimensions has been proposed to
accommodate globular protein domains up to a molecular weight
of approximately 15 kDa [87]. It is hypothesized that the hydroTable
1
Relationship between disulﬁde bond pattern and folding dependence on DsbC. Table summarizing the drop in either activity or yield of proteins when expressed in cells lacking
dsbC. Results are shown as % activity or yield relative to wild type (dsbC+) cells. Schematic representation of the disulﬁde bond pattern depicted as yellow balls from amino
terminus (left) to carboxyl terminus (right). Shaded boxes indicate proteins with consecutive disulﬁde bond patterns which are not dependent on DsbC.
Protein Disulﬁde bond pattern Relative activity/yield in DdsbC (%) Ref.
PhoAAlkaline phosphatase 100 [153]
hGH Human growth hormone 100 [80]
Agp Glucose-1 phosphatase 100 [78]
Anti CD-18 Human fab 100 [80]
Hcp H. pylori cysteine-rich protein B 100 [79]
MGSA Melanocyte growth stimulating factor 50 [80]
IGF-I Interferon like growth factor I 50 [80]
MepA Murein hydrolase 25 [154]
BPTI Bovine pancreatic trypsin inhibitor 20 [76]
AppA Acid phosphatase 20 [78]
RBI Ragi bi-functional inhibitor 7 [84]
RNaseI Periplasmic nuclease 0 [154]
tPA Tissue plasminogen activator 0 [82]
Urokinase 0 [76]
244 M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251
phobic cleft of DsbC discriminates against interacting with correctly
folded proteins whose hydrophobic amino acids reside sheltered
within the core of the protein. Instead, DsbC may prefer
interacting with misfolded proteins whose hydrophobic core is exposed
due to mis-oxidation. Also, based on analysis of protein crystal
structures, correctly oxidized cysteine pairs are usually
inaccessible and buried within the protein while mis-oxidized
pairs may be exposed and accessible to DsbC.
DsbC’s hydrophobic cleft may assist folding of proteins, even
those that lack disulﬁde bonds altogether. For example, puriﬁed
DsbC helps refold in vitro three different substrate proteins: nondisulﬁde-bonded
D-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), lysozyme [88] and single-chain antibody fragments
[89]. This chaperone property of DsbC is dependent on the dimerization
of DsbC [90] and is independent of its redox-active cysteines.
Other proteins with similar hydrophobic clefts are known to
be folding chaperones, such as maltose binding protein [91] and
trigger factor [92]. However, no direct evidence has thus far shown
the role of the hydrophobic cleft in the ability of DsbC to bind and
refold misfolded proteins.
Although disulﬁde bond isomerization has been studied in vitro
using the model protein BPTI [93], the in vivo mechanism remains
in dispute. Currently there are two in vivo models for how DsbC
facilitates disulﬁde bond isomerization. (i) The isomerization model
and (ii) the cycles of oxidation/reduction model. Both models
have the same initial step of a reduced DsbC attacking a disulﬁde
bond within the protein and forming a mixed-disulﬁde-bonded
complex. The subsequent steps of resolving this complex are different
for the two different mechanisms (Fig. 3).
(i) The ﬁrst isomerization model is based on the observation
that DsbC can shufﬂe the disulﬁde bonds of a mis-oxidized protein
in vitro within the appropriate redox buffer in the absence of any
other accessory protein [93]. In this model, the mixed-disulﬁdebonded
DsbC-protein complex is resolved by a nucleophilic attack
from another disulﬁde bond within the mis-oxidized protein,
resulting in the shufﬂing of disulﬁde bonds. The isomerization of
disulﬁde bonds is guided by the substrate protein’s folding landscape
and results in the target protein’s most thermodynamically
stable, fully folded and oxidized state. The ﬁnal nucleophilic attack
would return DsbC back to its reduced state.
(ii) The second oxidation/reduction model is based on the
observation that DsbC displays strong in vitro reductase activity,
equivalent to the reductase activity of cytoplasmic thioredoxin
[94]. Should the mixed-disulﬁde-bonded complex between DsbC
and the mis-oxidized protein not be resolved by a nucleophilic attack
from the substrate protein, then DsbC can resolve itself by
reducing the disulﬁde bond, leaving it in an oxidized state. This
would result in the reduction of the wrong disulﬁde bond, allowing
for subsequent re-oxidation by DsbA. This separation by reduction
of the protein-DsbC complex is essential for DsbC to resolve itself
from a non-productive complex, when the protein is too misfolded
to be isomerized. Otherwise, one could imagine titration of functional
DsbC from the periplasm, under conditions where there is
a large number of misfolded proteins. This model was shown to
be plausible in vivo; DsbC can be complemented in vivo by expression
of a disulﬁde bond reductase. Correct folding and oxidation of
a natural substrate of DsbC was obtained, in the absence of any
detectable disulﬁde bond isomerase activity [49].
Fig. 3. The two in vivo models of disulﬁde bond isomerization. rA reduced protein (PRED) is mis-oxidized (PMIS-OXI) by DsbA. The misfolded protein is recognized by DsbC
forming mixed-disulﬁde-bonded complex. s In the reducing pathway, t-uDsbC reduces the wrongly formed disulﬁde, v allowing for DsbA to have another chance at
correctly oxidizing the protein. w In the isomerization pathway, DsbC acts as a true isomerase allowing for the shufﬂing of the disulﬁde bonds, x resulting in no change in the
redox state of DsbC or the substrate protein.
M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251 245
Alternative mechanisms of periplasmic disulﬁde bond formation
Successful folding of a recombinant protein to high yields is
both highly unpredictable and dependent on the expression conditions
and substrate protein. It is therefore advantageous to have
multiple strain backgrounds that promote oxidative folding, enabling
the researcher to ﬁnd the optimum system for expressing
active correctly folded protein. This section will brieﬂy review
the various mutant strains that allow for the production of disulﬁde-bonded
proteins in the periplasm of E. coli.
Alternative oxidoreductases
Elucidation of the periplasmic disulﬁde bond forming machinery
in E. coli has allowed researchers to circumvent the native
dsb pathway in search of novel mechanisms of forming disulﬁde
bonds. Some genetic selections for chromosomal mutations that
restored function in the dsb pathway resulted in simple up regulation
of other dsb genes [95,96]. Here, we describe other selections
systems that sought to replace the function of a Dsb protein with
another oxidoreductase either from E. coli or for from other
organisms.
One obvious candidate that might replace DsbC is E. coli’s second
periplasmic oxidoreductase DsbG. DsbG is homologous to
DsbC with 49% amino acid sequence similarity and 30% identity.
Its crystal structure reveals a very similar architecture to DsbC
but its protein binding cleft is larger and less hydrophobic [97].
Even though DsbG can catalyze disulﬁde bond isomerization
in vitro [98] and has signiﬁcant chaperone activity [99], no
in vivo substrate that requires its disulﬁde bond isomerase activity
has been identiﬁed. When over-expressed from a plasmid, DsbG
can complement a dsbC null strain by assisting the folding of BPTI
[100] and AppA and by activating P1 lysozyme via intramolecular
thiol-disulﬁde isomerization (141) (data not shown). However,
DsbG’s natural role is likely to act as a reductase, restoring cysteines
that have been oxidized to sulfenic acid back to their reduced
thiolate state [101]. Due to the differences in substrate speciﬁcities
between DsbC and DsbG, highly expressed DsbG in theory may assist
in the folding of disulﬁde-bonded proteins which DsbC fails to
fold. This difference between DsbC and DsbG was used to isolate
mutant DsbG’s that could complement the copper sensitivity of
dsbC null strains [102].
Scientists have also successfully identiﬁed eukaryotic proteins
that can complement DsbA function. In eukaryotes, disulﬁde bond
formation and isomerization is catalyzed by a single enzyme called
protein disulﬁde bond isomerase (PDI) [103]. Overexpression of rat
PDI in the periplasm of E. coli complements a dsbA null strain,
increasing the yield of BPTI sixfold [104]. Overexpression of human
PDI also complements a dsbA strain, increasing the yields of Fab
fragment [105] and pectate lyase C ﬁvefold [106]. Thus, expression
of PDI in E. coli may improve the yields of certain substrate
proteins.
Engineered oxidoreductases
In addition to using native oxidoreductases, attempts have been
made to engineer and select mutants of various oxidoreductases to
replace Dsb protein function. Here we describe several of the alternative
proteins discovered.
Cytoplasmic oxidoreductases have also been shown to functionally
replace Dsb function. For example, when exported to the periplasm,
the cytoplasmic thioredoxin TrxA was functionally
converted from a disulﬁde bond reductase to an oxidase [16]. However,
periplasmic TrxA in a dsbA- strain was able to oxidize the
alkaline phosphatase (PhoA) such that PhoA activity was only
22% of wild type levels. Further optimization identiﬁed a mutant,
TrxA_G74S [50], that interacted better with DsbB [49], restoring
PhoA activity to levels similar to wild type.
In an attempt to identify de novo pathways and circumvent the
DsbA/DsbB oxidative pathway, Masip et al. selected an exported
mutant thioredoxin that restored a double null dsbA dsbB phenotype
[107]. The selection scheme required the mutant thioredoxin
to be folded in the oxidizing cytoplasm of a trxB gor strain (see below
for more details on a trxB gor strain) and be exported to the
periplasm via the TAT pathway. Surprisingly, the mutant thioredoxin
formed a dimeric iron-sulfur cluster protein and was able
to oxidize periplasmic PhoA independent of dsbA and dsbB. However,
restoration of the oxidizing pathway yielded only 40% of wild
type levels of PhoA. Another approach to circumventing the dependence
on DsbA/DsbB for the oxidative folding of proteins was to
export the E. coli glutaredoxin Grx3 to the periplasm [108]. Expression
of periplasmic Grx3 resulted in PhoA activity at approximately
60% of wild type levels. Intriguingly, disulﬁde bond formation by
periplasmic Grx3 was dependent on the cytoplasmic production
of glutathione. Exogenously added glutathione increased the level
of PhoA activity to 75% of wild type cells in the presence of periplasmic
Grx3.
Another approach utilized the architecture of DsbC as a scaffold
to engineer novel disulﬁde bond isomerases. DsbC is a dimeric protein
where each monomer contains a thioredoxin domain and a
dimerization domain. Dimeric DsbC cannot act as an oxidant as it
does not interact with DsbB due to steric hindrance. Selection of
mutant DsbC proteins that can complement a dsbA strain resulted
in monomeric DsbC that contain mutations in the dimerization domain
[109]. The monomeric DsbC interact with DsbB and oxidize
PhoA activity to 50% of wild type levels. Alternatively, deleting
amino acid residues from the linker domain of DsbC also allows
it to interact with DsbB, and thus function as an oxidase [110]. Finally,
in an attempt to replicate the architecture of DsbC, Arredondo
et al. engineered novel chimeric oxidoreductases utilizing the
dimerization domain of proline cis/trans isomerase FkpA, fused to
DsbA [111]. These chimeric constructs were able to complement
the dependence of vtPA on DsbC up to 80% of wild type (DsbA+/
DsbB+) levels.
The two amino acid residues in between the two cysteines of
the active site (CxxC) are known to act as ‘rheostats’, calibrating
the activity of an oxidoreductase from a reductase to an oxidase
[18]. Changing these active site amino acids has signiﬁcant effects
on the activity of TrxA [19], DsbA [48], DsbB [112] and PDI [113].
More importantly, for the purposes of protein production, DsbC
variants that contain alterations in the dipeptide sequence of its
active site residues, increase the yield of mouse urokinase up to
ﬁvefold [114].
Fusion partners
In general, DsbA interacts with largely unfolded polypeptides as
they are entering the periplasm. A hydrophobic groove near the active
site of DsbA has been proposed to be its substrate binding cleft
[115] and DsbA displays some chaperone activity in vitro [116]. The
ability of PDI, DsbA and DsbC to bind to unstructured polypeptides
via their hydrophobic binding clefts has been used to improve protein
folding and disulﬁde bond formation by constructing fusion
substrate proteins to either DsbA [117,118], DsbC [83], PDI [119–
121] or TrxA [122,123]. Plasmid expression vectors which permit
cloning of one’s gene of interest as a fusion protein to DsbA
(pET-39), DsbC (pET-40) or TrxA (pET-32) are currently available
from Novagen.
These results highlight the interchanging roles of disulﬁde bond
oxidoreductases from reductases to isomerases to oxidases. The
function of one oxidoreductase can be readily converted to another
by manipulating the compartments it is expressed in and by introducing
subtle mutations that have profound effects on its activity,
architecture and interacting protein partners.
246 M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251
Cytoplasmic production
The cytoplasm of wild type E. coli is not amenable to the production
of disulﬁde-bonded proteins [124]. Although disulﬁde
bonds may form transiently, they are quickly reduced by the
numerous reductases and small molecule reductants present in
the cytoplasm. This ‘reducing environment’ is maintained by the
glutaredoxin and thioredoxin pathways (Fig. 4) [125]. Both pathways
receive their reducing potential from the cytoplasmic pool
of NADPH. In the case of the thioredoxin pathway, thioredoxin
reductase (trxB) maintains two thioredoxins in their reduced state,
Trx1 (trxA) and Trx2 (trxC) [126]. These thioredoxins in turn reduce
cytoplasmic proteins which might form disulﬁde bonds (either by
oxidative damage or through their catalytic activity) back to their
thiolate state.
An alternative pathway for maintaining a reducing cytoplasm is
the glutaredoxin pathway. Glutathione reductase reduces two enzymes,
GshA and GshB, which are responsible for synthesizing reduced
glutathione (GSH). Glutathione is a small tripeptide (L-cglutamyl-L-cysteinylglycine)
whose primary role is to act as the
major component of the cytoplasmic redox buffer. At intracellular
concentrations approaching 5 mM, most glutathione is reduced
and not in its disulﬁde-bonded dimeric form, GSSG. In the cytoplasm
of E. coli, the ratio of GSSG:GSH is 1:200 [127]. GSH also directly
reduces disulﬁde-bonded proteins, forming a GSH-protein
complex. The complex is resolved by one of several glutaredoxins
(Grx1, Grx2 and Grx3) that reduce the protein and oxidize glutaredoxin.
The oxidized glutaredoxin is recycled back to its active reduced
state by GSH [128].
Ribonucleotide reductase (RNR) is an essential protein, whose
cysteines become oxidized during its catalytic cycle and requires
either the thioredoxin or the glutaredoxin pathway to recycle it
back to its active reduced state [129,130]. Since the reducing
capacity of either the thioredoxin or the glutaredoxin pathway is
sufﬁcient to reduce RNR, genetic disruption of either pathway is
tolerated by E. coli. However, cells are not viable when both pathways
are disrupted. Deletion of both trxB and gor leads to lethality
but can be suppressed by a mutation in the peroxidase AhpC. This
mutant AhpC⁄
has lost its peroxidase activity but has gained disulﬁde
bond reductase activity [131]. AhpC⁄
had increased capacity to
resolve GSH-Grx complexes in vitro; presumably, this increased
capacity results in sufﬁcient amounts of reduced Grx1 in vivo to restore
viability [132]. However, in the absence of thioredoxin reductase
(trxB), the two thioredoxins in E. coli (Trx1 and Trx2)
accumulate in their oxidized forms enabling them to act as disulﬁde
bond formation catalysts in a reversal of their normal function
[123]. This ﬁnal strain FÅ113 (trxB, gor, ahpC⁄
) has the remarkable
capacity to catalyze disulﬁde bond formation in the cytoplasm
[133]. FÅ113 is commercially available under the name Origami
(Novagen) and has been used to express numerous disulﬁdebonded
proteins in the cytoplasm [134–139].
One drawback to using strains such as FÅ113 for the production
of disulﬁde-bonded proteins is the lack of disulﬁde bond isomerization.
If a protein is mis-oxidized in the cytoplasm of FÅ113, the
protein remains mis-oxidized since the disulﬁde bond isomerase
DsbC resides in the periplasm. Co-expression of DsbC in the cytoplasm
can greatly enhance the amount of correctly oxidized proteins
[85,86,133,140]. This is most likely due to DsbC’s disulﬁde
bond isomerase activity and not due to its weak oxidase activity
[93,141], as DsbC is mostly in its reduced state in the cytoplasm
of FÅ113 [133]. To generate an oxidative strain with disulﬁde bond
isomerase capacity, a new protein expression strain named SHufﬂe
(New England Biolabs) was recently engineered that expresses
DsbC in the cytoplasm of a trxB, gor, ahpC⁄
strain. Since SHufﬂe’s recent
commercial release, it has been shown to successfully express
a membrane protein U24 from human herpes virus [142].
Whether a protein requires the corrective action of DsbC for its
oxidative folding depends strongly on the mechanism of disulﬁde
bond formation. Disulﬁde bond formation in the periplasm is profoundly
different than in the cytoplasm. In the periplasm, the
majority of disulﬁde bond formation is catalyzed by DsbA which
has been shown to occur co-translocationally as the unfolded
polypeptide enters the periplasm [17]. This strongly favors the oxidation
of cysteines in a consecutive manner (Table 1). In comparison,
the mechanism and timing of disulﬁde bond formation in the
cytoplasm of trxB, gor cells has not been studied in detail. Unlike
the periplasmic entrance of polypeptides, there is signiﬁcant folding
of the nascent chain within the ribosome exit channel and at
the exit pore in the cytoplasm [143]. The partial folding of the protein
may result in the formation of the correct disulﬁde bonds,
Fig. 4. Disulﬁde bond formation in the cytoplasm of SHufﬂe cells. Schematic representation of the redox pathways involved in the formation of disulﬁde bonds, in the
cytoplasm of SHufﬂe cells. Disruption of the thioredoxin pathway (TrxB) and the glutaredoxin pathway (Gor) is lethal. Selection of trxB, gor suppressor resulted in mutant
AhpC⁄
which lost the ability to reduce peroxides but gained the ability to reduce Grx1. (A) Reduced Grx1 in turn can catalyze the reduction of proteins. Accumulation of
oxidized thioredoxins such as Trx1 catalyzes the formation of disulﬁde bonds. (B) If the protein is mis-oxidized, it is isomerized to its native correctly folded state (C) by
cytoplasmic DsbC. Disabled protein interactions are represented by dotted lines. For simplicity, other reductases (Grx2, Grx3 and Trx2) are omitted and only the redox state of
cysteines (yellow balls) of Grx1 and Trx1 are indicated (oxidized = yellow balls are joined; reduced = ball). (For interpretation of the references in colour in this ﬁgure legend,
the reader is referred to the web version of this article.)
M. Berkmen / Protein Expression and Puriﬁcation 82 (2012) 240–251 247
making the protein less dependent on DsbC. This may explain why
urokinase activity is fully dependent on DsbC when expressed in
the periplasm but only 50% dependent when expressed in the cytoplasm
(data not shown). Furthermore, the cytoplasm contains signiﬁcant
amounts of glutathione, which is involved in oxidative
folding [144], while the concentration and redox state of periplasmic
glutathione is signiﬁcantly different [108,145]. These differences
between the two compartments can have a signiﬁcant
effect on the ﬁnal yields of a protein. For example, the yield of collagen
prolyl 4-hydrolase was 20 times higher in the cytoplasm of
FÅ113 (Origami) than in the periplasm of the corresponding BL21
wild type strain and 10 times better than when expressed in insect
cells [146].
Our current view on the mechanism of disulﬁde bond formation
in the cytoplasm of FÅ113 is incomplete. For example in FÅ113
(trxB, gor, ahpC⁄
), it is unknown which of the two thioredoxins
are involved in the oxidation of proteins. Trx1 and Trx2 share
66% similarity and 29% identity in their amino acid sequence and
have very similar functions [123,147,148]. Trx1 levels are tenfold
higher than Trx2 levels. However, Trx2 is under the regulation of
OxyR and is expressed at much higher levels under oxidative stress
conditions. Since under oxidative conditions, Trx2 remains reduced
in comparison to Trx1, Trx1 may be specialized towards protection
against oxidative damage. This may explain why 75% of the cytoplasmic
PhoA activity in a trxB mutant strain is attributable to
Trx1 while only 25% is due to Trx2 [123].
Alternative mechanisms of cytoplasmic disulﬁde bond formation
Secretion of a protein to the periplasm is problematic and may
not result in the formation of the correct disulﬁde bonds [149].
Cytoplasmic oxidative folding of one’s protein of interest in a trxB,
gor strain is therefore an attractive solution. However, not all proteins
expressed in a trxB, gor strain fold and form soluble active
protein. For proteins that fail to fold properly in the cytoplasm of
a trxB, gor strain, there are alternative strains to try. Recently Ruddock
and colleagues have shown that the co-expression of the
mitochondrial inner-membrane FAD-dependent sulfhydryl oxidase
Erv1p can promote the de novo formation of disulﬁde bonds in the
cytoplasm [150]. While co-expression of Erv1p did not increase the
yield of PhoA or AppA in the cytoplasm of a trxB, gor strain, remarkably
Erv1p increased the yield of PhoA in wild type BL21 cells.
Intriguingly, co-expression of Erv1p along with DsbC resulted in
a threefold increase in the activity of AppA, a natural substrate of
DsbC [78]. Thus, disulﬁde bond formation is not purely a result
of the redox environment (i.e., the oxidizing periplasm) but a result
of the presence of the appropriate disulﬁde bond-forming catalysts.
In the case of the trxB gor strain, the oxidized thioredoxins
can catalyze the formation of disulﬁde bonds, even when there is
sufﬁcient reducing power within the cytoplasm to reduce other
essential proteins. In the case of Erv1p, the simple cycling of reduced
Erv1p by molecular oxygen is sufﬁcient to catalyze the oxidation
of both PhoA and AppA, while both the thioredoxin and
glutaredoxin pathways are active. Further improvements to the
yields were achieved by pre-expressing Erv1p, prior to the expression
of the substrate proteins [151]. The utility of this system was
recently demonstrated in the production of camel antibodies [152].
Co-expression of Erv1p along with DsbC or PDI resulted in 10-fold
enhancement of yield, reaching yields of tens of milligrams.
Conclusions
Much progress has been achieved since the days of Anﬁnsen.
Today we have a much more in-depth understanding of the processes
that govern protein folding. E. coli remains a useful model
organism for the study of disulﬁde bond formation, both in the periplasm
and the cytoplasm. Combinations of genetic engineering
and fortuitous discoveries have led to the discovery of novel mechanisms
of disulﬁde bond formation. These, in turn, have allowed
scientists to engineer new strains and develop novel systems for
producing disulﬁde-bonded proteins. The most recent ﬁndings of
forming disulﬁde-bonded proteins within the reducing cytoplasmic
environment of wild type cells have broken our chains to last
century’s dogmas and opened new horizons of what is possible.
Expanding our studies to other organisms will undoubtedly bring
forth new insights and mechanisms of disulﬁde bond formation
within living cells.
Acknowledgements
I would like to thank Jon Beckwith, Chris Taron, Melanie
Berkmen, Paul Riggs and Iris Walker for the critical reading of
the manuscript.
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