Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

A PRACTICAL GUIDE TO PROTEIN EXPRESSION AND REFOLDING

FROM INCLUSION BODIES

Lisa D. Cabrita, Michelle K.M. Chow and Stephen P. Bottomley

This is an edited version of:

“Protein Expression and Refolding – a practical guide to getting the most out of inclusion
bodies”, Cabrita and Bottomley, 2004, Biotechnology Annual Review, 10:31-50.

If referring to anything in this document, please cite the above paper.

Contents

1. Introduction
2. Recombinant protein expression in E.coli
2.1 Promotors and fusion partners
2.2 Codon bias, disulfide bond formation and E.coli strains
2.3 Cell-free expression
3. Preparation and solubilization of inclusion bodies
4.
5. Refolding
5.1 Protein concentration during refolding
5.2 The refolding buffer
5.3 Disulfide bond formation
5.4 Refolding methods
5.5 Folding aids
5.6 Folding screens
6. Analysis of refolded protein
7. Conclusion
Figure 1
Table 1
Table 2
Table 3
References

1
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
1. INTRODUCTION
One of most significant aspects of biomedical
and scientific research in the post-genomic
era is the ability to rapidly express and purify
a target protein. With an immense amount of
sequence data available, all over the world,
structural genomic projects are being
undertaken in which a vast array of proteins
are expressed, purified and structurally
characterised. In addition to these high-
throughput approaches, both academic and
industrial laboratories are expressing proteins
of interest for structural, functional and
therapeutic investigations. The general
course for this process involves rapid cloning
of target genes, followed by recombinant
expression of the protein, usually in the host
E.coli.
Currently, E.coli remains the dominant
expression host for recombinant proteins,
due to its advantages such as ease of
handling, cost effectiveness and high
success rates. However, it does have some
disadvantages, for example, the inability to
perform many post-translational modifications
and frequent deposition of the expressed
protein product into insoluble inclusion
bodies. Inclusion body formation is often an
undesired event, and a number of
approaches can be utilized to obtain soluble
protein. On the other hand, inclusion body
formation does not necessarily have to be a
dead-end process. Sometimes harvesting of
proteins from inclusion bodies can prove to
be very fruitful and may present a valid option
for successful purification (Figure 1). In
recent years, there has been an increase in
products and protocols which are generally
applicable to the inclusion body issue. New
strains of E.coli and fusion partners are
available, which offer a range of options to
minimize, or maximize, inclusion body
formation. Furthermore, a more rational
approach to protein refolding has been
established, which allows the user to
determine very quickly whether refolding from
inclusion bodies is a viable option.
2
2. RECOMBINANT PROTEIN EXPRESSION
IN E.COLI
The expression of recombinant proteins
within E.coli is affected by several factors.
These include, but are not limited to: plasmid
copy number, mRNA stability, upstream
elements, temperature and codon usage. The
expression of an uncharacterized protein
often depends largely on a combination of
these and other factors, and success in
protein expression is often difficult to predict.
However, over the years numerous advances
have been made to improve both expression
and solubility. This has been made possible
through the development of novel tags, fusion
partners, and vector systems, such that the
choices for recombinant expression in E.coli
are continually expanding.
2.1 Promoters and fusion partners
In regulating protein expression, the choice of
promoter and/or vector system is important,
as one system may be more suited to a given
target protein than another. Several
promoters are available (Table 1) - probably
the most well-known variety is the T7-derived
promoter, as found in the pET vectors
(Novagen). These IPTG-inducible promoters
have in the past been associated with “leaky”,
or premature, expression prior to induction,
which is problematic for proteins which are
toxic to cells. However, this issue has been
addressed with co-transformed plasmids
pLysS and pLysE which can act as strong
repressors. Aside from this issue, the T7 is a
strong promoter which enables high level
expression of target proteins. Overexpression
can also lead to the formation of intracellular
inclusion bodies, sometimes in far greater
excess than the soluble protein. This
insoluble-soluble ratio can sometimes be
adjusted by altering the expression time,
induction temperature and/or IPTG levels.
The T7-based vectors have previously
included affinity tags to facilitate the detection
and purification of proteins, although they are
also now often designed to include other
features. Of interest are ‘fusion partners’ that
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
in general, ‘fuse’ the protein of interest with
another, to circumvent the solubility issue and
also aid in purification. The classic GST
fusion system has been implemented
successfully for a large number of proteins [1-
4], however more recently, a number of other
alternative options have also been developed
(Table 2 and [5]). One example is the 42 kDa
maltose binding protein (MBP), which has
been used successfully to increase the
solubility of a range of targets [6-8] and has
been shown to have little effect during
crystallization - this is a great advantage, as
fusion tags generally need to be removed to
aid in crystallography. A similar tag is Nus A
(Novagen), a 54 kDa protein, which was
identified as being the most soluble protein
out of a pool of almost 4000 E.coli proteins
[9]. As well as assisting in folding, some tags,
such as the ~15 amino-acid S-tag (Novagen),
for example can be used to detect, quantitate
and purify soluble protein [10].
2.2 Codon bias, disulfide bond formation
and E.coli strains
Poor expression of a target protein may also
be associated with codon bias – that is,
codons used infrequently in the prokaryotic
system. The Rosetta (Novagen) and BL21-
CodonPlus strains (Stratagene) have the
advantage of co-expressing plasmids that
code for the rare tRNA’s and have been used
with success for a number of proteins [11-13].
Moreover, the development of E.coli that
accommodates disulfide bond formation can
also improve the yields of certain targets. The
AD494(DE3) strain (Novagen), which has a
single mutation in the thioredoxin reductase
gene, has enabled the production of a range
of proteins [14-16]. The Origami strain
(Novagen), however, combines a double
mutation in both the thioredoxin reductase
and the glutathione reductase gene, hence
providing a more favourable oxidizing
environment within the cytoplasm.
Furthermore, the recent Rosetta-Gami strain
(Novagen) is a powerful combination of the
Rosetta and Origami strains, encompassing
rare codon usage and disulfide bond
3
formation. This strain may be particularly
useful for eukaryotic or intracellular proteins
which typically display alternative codon
usage and require disulfide bond formation,
respectively.
2.3 Cell-free expression
Cell free expression (also known as ‘in vitro
transcription-translation’) has been an
invaluable tool for many years. It exploits the
E.coli, wheat germ and rabbit reticulocyte
systems to generate protein and is
increasingly seen as a viable alternative to
other expression systems for recalcitrant
proteins. The technology itself was developed
some 30 years ago [17], however, more
recently been subject to several significant
developments [18,19]. These include the
‘continuous-flow’ system whereby amino
acids and energy sources are supplied into a
reaction chamber, while synthesized proteins
and used substrates are removed using an
ultrafiltration membrane [18], and the
“semicontinuous’ method, involving the use of
two chambers separated by a dialysis
membrane [19,20]. Roche also now offers a
in vitro transcription-translation ‘Rapid
Translation System’, which has been
successful for a number of targets, and can
also incorporate molecular chaperones,
detergents and other additives to assist in
folding.
Although not as efficient as the E.coli
system, the cell free system based upon
wheatgerm has also been used [21-23].
Being a eukaryotic system, it has been
explored as an alternative, particularly for the
production of proteins that may not otherwise
express in E.coli. Cell-free protein expression
has been limited in the past by low efficiency,
membrane clogging and consequent
questionable reproducibility. However, in light
of recent advancements in the technology,
continued improvements may encourage
more frequent use and in the future it may
emerge as an increasingly viable alternative
to the traditional E.coli expression system.
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
3. PREPARATION AND SOLUBILIZATION
OF INCLUSION BODIES
Inclusion bodies are dense amorphous
aggregates of misfolded protein. They are
generally formed if a protein has a high
propensity to misfold and aggregate, or if the
cellular protein production machinery is
overwhelmed and unable to operate
efficiently. The formation of inclusion bodies
can sometimes be especially advantageous,
especially if the protein is toxic to the cell, or
susceptible to proteolytic attack.
Inclusion bodies are easily identifiable,
with a morphology similar to strings or
clusters [24], and their isolation from other
cellular components can be easily achieved.
Inclusion bodies are contained within the
insoluble fraction yielded from cell lysis and
subsequent centrifugation, and it is generally
accepted that they may be composed of 40-
90% target protein. Successful renaturation
depends on the purity of inclusion bodies, as
proteinaceous contaminants can decrease
the efficiency of renaturation, presumably as
such contaminants can promote co-
aggregation [25]. Inclusion bodies are
therefore best washed and centrifuged a
several times in buffer containing detergents
such as Triton X-100 (0.1-4% (v/v)), sodium
deoxycholate (2% (w/v)), sarkosyl, or even
low concentrations (0.5-1 M) of denaturants
such as guanidine hydrochloride (GdnHCl) or
urea. Such additives remove cellular
contaminants that adsorb onto the
hydrophobic inclusion bodies.
Following washing, inclusion bodies are
then solubilized, usually with 4-6 M GdnHCl
or 8 M urea. GdnHCl is a stronger
denaturant, while during prolonged
incubations at alkaline pH urea can suffer
from the formation of isocyanate ions which
can modify amino acid side chains [26,27].
Aside from denaturants, other unfolding
alternatives include detergents such as
sarkosyl [28,29], SDS [30] and alkaline pH
[31,32]. It is important that during unfolding
any disulfide bonds present are also reduced
– this is usually achieved with β-
mercaptoethanol or DTT (5-100 mM). The
4
need for disulfide bond reduction is
highlighted by a pivotal study which
demonstrated that these bonds persist in high
denaturant concentrations in the absence of
reducing agents [33]. Usually solubilization
can be performed in any buffer that is
compatible with the protein of interested, for
example Tris, Hepes or phosphate. Generally
a neutral pH (7-8) is suitable for
solubilization.
Upon solubilization of inclusion bodies,
ample incubation time should be allowed for
complete unfolding to occur. Incubation at
either room temperature or 30°C for 1-4
hours is generally sufficient, whilst some
studies have opted for 16-24 hours at 4°C.
Sometimes inclusion bodies may be difficult
to solubilize and therefore some agitation or
increased temperature may be required.
Sometimes inclusion bodies are purified
further by column chromatography such as
ion exchange, size exclusion or metal affinity
conducted under denaturing conditions [34-
36]. This has been seen to enrich the
proportion of monodispered protein which
can increase the yield of the refolded target
protein [37]. More recently, it has been shown
that inclusion bodies could be directly purified
by gel filtration only using a macroporous
medium (eg. 4% agarose) coupled with a
French press to reduce cell debris [38].
4. REFOLDING
The renaturation process aims to effectively
remove the denaturant and thiol reagents and
allow the protein to refold. The refolding
process is a competing reaction with
misfolding and aggregation events and its
success depends on a number of factors.
Studies have shown that for many proteins,
especially those larger than 150 amino acids,
folding involves the formation of an
intermediate species, often resembling a
‘molten globule’. The molten globule
ensemble can represents a branch point in
the folding pathway where it may also lead to
misfolding and aggregation, as this species
contains some secondary structure, but little
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
tertiary structure and hydrophobic patches
normally buried within the protein are
exposed to solvent [39]. Under appropriate
conditions these regions can promote
aberrant interactions that may lead to
aggregation. Therefore it is important to
minimize the aggregation reaction. Several
variables play important roles in this process,
including final concentration of the protein to
be refolded, the components of the refolding
buffer and the method of refolding.
4.1 Protein concentration during refolding
The amount of protein refolded will impact the
yield of protein obtained. Folding competes
with aggregation, and therefore it is generally
accepted that refolding at low protein
concentrations (10-100µg/ml) is the most
successful approach. There have been
situations where proteins have been refolded
in high concentrations of up to 5 mg/ml [40-
42], albeit in low concentrations of
denaturant, however, generally speaking, the
lower the final protein concentration during
refolding, the greater the efficiency of the
process.
4.2 The refolding buffer
Components of a refolding buffer vary widely,
depending on the protein of interest.
Parameters such as pH, ionic strength, redox
conditions and the presence of ligands can all
influence the outcome of refolding. Most
commonly Tris or Hepes-based buffers at
neutral pH with 50-500 mM NaCl are used,
however again this depends on the target
protein. Numerous additives can also be
included, with varying success with numerous
targets. These include detergents, polar
additives, weak chaotrophs, osmolytes and
cations (Table 3). Such additives may either
act as stabilizers (which stabilize the native
state or solubilize intermediate forms), or
promote correct folding by preventing
aggregation. If the target protein is prone to
proteolysis, a common approach is to include
proteinase inhibitors (eg. PMSF, aprotinin,
leupeptin) in the refolding buffer.
5
4.3 Disulfide bond formation
A redox system during renaturation is
required for the correct formation of proteins
with native disulfide bonds. The combination
of reduced and oxidized forms of redox
agents are generally used in molar ratios
from 1:1 up to 10:1. Glutathione
(GSH/GSSG) is a common reagent, however,
other alternatives may include a combination
of cysteine and cystine, or DTT/oxidized
glutathione. While molecular oxygen in the air
is suitable and sufficient to promote disulfide
bond formation, a redox system accelerates
the shuffling of disulfide bonds. Trace
amounts of metal ions and slightly alkaline
pH (8-9) may also be included to catalyze the
redox reaction. EDTA can be beneficial to
minimize the effects of oxygen oxidizing
thiols. The time required for disulfide bond
shuffling varies between proteins, anywhere
from 2 hours to 150 hours. 16-48 hours is
common for efficient disulfide shuffling,
however, the exact time must be determined
empirically.
4.4 Refolding methods
Several different methods can be used to
refold proteins. These include dialysis,
dilution and column chromatography
techniques. The method selected depends on
the propensity of the protein to aggregate and
the kinetics of refolding. The temperature at
which refolding is performed may vary,
although generally, in order minimize
aggregation, 4°C is best.
Dilution
Refolding by dilution can be described as
‘rapid’ or ‘slow’. In rapid dilution, the
denatured protein is delivered to the refolding
buffer such that in a very short time period,
the concentrations of both the protein and
denaturant decrease rapidly. For example, if
the protein was denatured in 8 M urea and
diluted 10-fold into buffer, the final
concentration of denaturant is 0.08 M. The
aim of dilution is to remove the denaturant, so
its final concentration should be low. There
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
are instances, however, where refolding into
low molar concentrations of denaturant (1-2
M) aids in the solubility of the protein.
Rapid dilution has been used successfully
for a range of proteins [43-46], however, the
time period used dilution may be detrimental
for some proteins, especially if they usually
refold over a relatively long time period of
minutes to hours. Forcing the protein to adopt
its conformation in a short time frame may
increase its chances of misfolding and
aggregation. Slow dilution is an alternative
method, as it is a more gentle approach and
dramatically decreases the effective
concentration of the refolding protein. The
‘dropwise’ or ‘pulsed dilution’ method involves
the solubilized inclusion bodies being
delivered very slowly to the refolding buffer
(using a pump) and has been successfully
used for some proteins [36,47].
Dialysis
In this method, the concentrated denatured
protein is dialyzed against a refolding buffer,
such that the concentration of denaturant
decreases with buffer exchange. This slow
removal of denaturant allows refolding to
occur. Unfortunately, the slow removal of
denaturant often results in the formation and
exposure of long lived intermediate species
over a time period and hence there may be
an increased propensity for the protein to
aggregate. A variation of one-step dialysis,
where protein is dialyzed against refolding
buffer containing no denaturant, is the use of
a series of refolding buffers with decreasing
denaturant concentrations. By dialyzing in a
step-wise fashion (usually 1-3 progressive
lower denaturant concentrations), this allows
an equilibrium to be established. Once again,
whilst refolding is more controlled in this
environment, long-live intermediate species
can still be problematic.
On-column refolding
On-column refolding has been applied to
several proteins with success and offers an
alternative where other methods may not be
6
fruitful. If a protein has a hexa-histidine tag, it
can be immobilized on a Ni2+ affinity column
in its denatured state. By applying buffers
with a decreasing concentration of
denaturant, either step-wise or by using a
gradient, the protein can be refolded and then
eluted [48,49].
Gel filtration is an alternative technique,
whereby the denatured protein is loaded onto
the column and refolds as it is passed
through with buffer [50,51]. A variation of this
is to equilibrate the column with a linear
gradient with decreasing denaturant
concentrations, such that the protein refolds
gradually [52]. Flow rates required for
successful on-column refolding vary – for
some proteins, slower flow rates improves
recovery [38], in other cases faster flow rates
are better [53].
On-column refolding methods have also
been improved by immobilizing common
‘foldases’ onto the column matrix and
exploiting them as a folding ‘platform’. This
has been demonstrated successfully with
GroEL [54,55]and DsbA/DsbC [56]. One
associated concern with on-column refolding
is the clogging of filters by aggregated
protein, however, carefully filtering or
centrifuging samples prior to loading onto the
column, and sensible choice of column matrix
can minimize these problems.
Other refolding techniques
Whilst a majority of proteins can be refolded
by classic dilution, dialysis or on-column
techniques, the potential development of
other refolding techniques is constantly being
explored by researchers. One recent foray
has been the use of reverse micelles, in
which the protein is trapped in a water-
sodium bis-2-ethylhexyl sulfosuccinate-
isooctane reverse micellar system [57]. This
was shown to result in the successful
recovery of monomeric protein where other
techniques had failed. Variation in the size of
the micelles enabled manipulation of the
degree of oligomerisation of the protein.
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.
4.5 Folding aids
Several molecular chaperones can be
included either in vivo or in vitro to aid folding.
The most well known E.coli chaperones
include GroEL-GroES, DnaK-DnaJ-GrpE
(Hsp70) and also ClpA/ClpB (Hsp100) which
have been used successfully in refolding
studies [58-61]. Whilst molecular chaperones
promote correct folding, foldases can
accelerate the process. The three known
types of foldases are 1) peptidyl prolyl
cis/trans isomerases (PPI’s), which arrange
prolines into their correct orientation [62], 2)
disulfide oxidoreductase (DsbA) and disulfide
isomerase (DsbC), which are found in E.coli
and promote disulfide bonds [63,64] and 3)
protein disulfide isomerase (PDI), a
eukaryotic protein which catalyses oxidation
and isomeration [65].
4.6 Folding screens
As finding the right conditions to refold a
protein can be a time-consuming process,
several commercial screens are available to
focus on potential conditions that return the
best yield of monomeric protein. One such kit
is Hampton Research’s FoldIt screen, which
employs a variety of additives, redox
conditions and pH. Similar kits are also
available through Novagen and Sigma-
Aldrich. These kits are the result of sparse
screens which have proven successful for the
refolding of a number of proteins [66-68]. The
commercially prepared kits enable a number
of refolding conditions to be sampled
simultaneously on a small scale, the aim
being to find suitable conditions which can be
up-scaled for batch refolding and purification.
An elegant example of using factorial
screens is demonstrated by a study involving
procathepsin S and cathepsin S [69]. An
initial screen used to identify folding
conditions targeted L-arginine and pH as two
key factors. While pH was important for both
proteins, arginine was more beneficial for
procathepsin only. Once these conditions
were established, a second screen based on
these findings was performeds to improve
7
yields. It was found that while procathepsin
folded best in the presence of detergent,
arginine and a redox system, cathepsin
required only glycerol and the redox couple.
This illustrates the powerful nature of the
factorial screen and also demonstrates how
seemingly similar proteins may have very
different requirements for refolding.
5. ANALYSIS OF REFOLDED PROTEIN
Once refolding is completed, it is necessary
to determine whether the process has yielded
folded or aggregated protein, and also
whether disulfide bonds (if any) have formed
correctly. Perhaps the simplest way to assess
the quality of the protein is by using a known
activity assay. While this is possible for some
proteins, a number of targets will have no
known function or structure, especially
considering the growing level of high
throughput production of proteins from
different genomes. Therefore, it is important
to develop other techniques to analyse the
nature of the protein.
Such techniques commonly include size-
exclusion chromatography and non-
denaturing PAGE, as well as circular
dichroism (CD) which reports on secondary
structure. Distinct features of a typical CD
scan report on the presence of α-helical, β-
sheet or random coil structure. Proteins will
generally be a combination of α-helical and β-
sheet content, while aggregated material may
produce a spectrum of predominately random
coil. Fluorescence techniques such as
dynamic light scattering [70], which
determines the average diameter of particles
in solution and lateral turbidimetry [6] can
also report the presence of aggregates [71],
as can ultracentrifugation [72] and electron
microscopy [73].
Assessing the nature of disulfide bonds
can be achieved by a number of methods.
Perhaps the most straightforward method is
the use of reducing and non-reducing gel
electrophoresis. In the absence of reducing
agent, there should be a defined ‘band shift’,
accompanied with any additional bands that
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

were record of linked domains. For a single

domain protein, a ‘laddering’ effect is a tell-
tale sign of intermolecular disulfide bond
formation. The use of thiol specific dyes
(eg.DTNB (Ellman’s reagent)) [74], mass
spectrometry [75] and gel electrophoresis
(‘cysteine counting’) [76] are all common
techniques that are also employed.

6. CONCLUSION
Handling inclusion bodies can be a time-
consuming and often frustrating trial-and
error process. Whilst fundamental principles
underpin the process, there are no clear rules
which apply to all proteins, and an approach
suitable for one protein maybe equally
inappropriate for another. Despite this,
working with inclusion bodies can be
beneficial, especially when it is impossible to
express reasonable quantities of soluble
protein. As more proteins are successfully
refolded, trends and patterns may become
clearer. This will aid the progression and
further development of biotechnological tools.

8
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

Figure 1: A schematic outline of the procedure for purification and refolding of proteins
from inclusion bodies
`

Cell Lysis
(eg.sonication, French press)

Centrifugation

Pellet (insoluble fraction) retained

Purification of Inclusion Bodies

Resuspension and centrifugation Chromatography (denaturing conditions)
Eg. Triton X-100, high salt, EDTA Eg. Gel Filtration, Ni-NTA

Solubilization of Inclusion Bodies

Example denaturants:
4-6 M GdnHCl 6-8 M Urea 0.5-2% SDS

Refolding
Dialysis On-column refolding Dilution
(direct or step) (linear or step-wise gradient) (step or drop-wise)

Post-refolding Analysis
Eg. Activity assay Circular Dichroism
Non-denaturing PAGE Size Exclusion Chromatography
Light-scattering Ultracentrifugation

9
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

Table 1: Examples of promoters often used in E.coli expression

Promotor type Example vector Inducing agent

T7 pET (Novagen) IPTG

T5 pQE (Qiagen) IPTG
trc pTrcHis (Invitrogen) IPTG
tac pMAL (New England Biolabs) IPTG
lac pTrip1Ex2 (Clontech) IPTG
Trp pLEX (Invitrogen) Tryptophan
araB PBAD (Invitrogen) L-arabinose
PL (λ) pKC30 Temperature shift (42°C)
phoA pBKIGF2B-A Phosphate
PLtetO-1 plP-PROTete-6xHN (Clontech) tetracycline

Table 2: Examples of fusion partners and tags which facilitate solubility and tracking
protein expression

Tag Size Location in relation to target

protein

Chloramphenicol acetyltransferase 24 kDa N terminus

Glutathione S-transferase (GST) 26 kDa N terminus
Maltose binding protein (MBP) 40 kDa N or C terminus
NusA 54 kDa N terminus
S-Tag 15 residues N or C terminus, or internal
Thioredoxin 11 kDa N terminus
Ubiquitin 76 residues N terminus
Z-domain (derived from Protein A) 58 residues N Terminus

10
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

Table 3: Examples of buffer additives which may be used to facilitate protein refolding

Additive Concentration used Effect

CHAPS 30 mM Detergent
EDTA 20 mM Chelator
Glycerol 10-50% Stabilizer
Guanidine HCl 0.1-1 M Chaotroph
L-arginine 0.4-0.5M Stabilizer
Lauroylsarcosine (sarkosyl) Up to 4 M Detergent
Lauryl maltoside 0.3 mM Detergent
MgCl2/CaCl2 2-10 mM Cation
NaCl/Ammonium sulfate 0.2-0.5 M Salt
Non-detergent sulfobetaine (NDSB) 256/201 Up to 1M Solubilizer
PEG 3350 Up to 0.05% (w/v) Osmolyte
SDS 0.1 % Detergent
Sodium citrate/sulfate 0.2-0.5 M Salt
Sucrose/glucose 0.4 M Stabilizer
TMAO 1-3 M Osmolyte
Tris 0.4-1 M Buffer
Triton X-100 0.1-1 % Detergent
Tween – 80 0.01 % Detergent
Urea 0.1-2 M Chaotroph

11
Protein Expression and Refolding from Inclusion Bodies, Cabrita et al.

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