MINIREVIEW
Concepts and tools to exploit the potential of bacterial
inclusion bodies in protein science and biotechnology
Pietro Gatti-Lafranconi
1,
*, Antonino Natalello
2,
*, Diletta Ami
2
, Silvia Maria Doglia
2
and Marina Lotti
2
1 Department of Biochemistry, University of Cambridge, UK
2 Department of Biotechnology and Biosciences, State University of Milano-Bicocca, Italy
Protein aggregation in the bacterial
cytoplasm: regulation, override and
effects
It is estimated that the global macromolecule concen-
tration in the Escherichia coli cytoplasm is around
200–400 gÆL
)1
and that macromolecules occupy 20–
30% of the total cytoplasmic volume [1,2]. Individual
proteins are represented at relatively low concentration
(nm to lm) but in the cytoplasm this translates into
the distance between any two molecules having the
same dimensions as proteins themselves [3]. Crowding
increases non-specific, attractive and electrostatic inter-
actions and modifies diffusion rates, with detrimental
effects on the behaviour of all macromolecules [4]. In

duction. As a result, in the bacterial cytoplasm several recombinant pro-
teins aggregate as insoluble inclusion bodies. The recent discovery that
aggregated proteins can retain native-like conformation and biological
activity has opened the way for a dramatic change in the means by which
intracellular aggregation is approached and exploited. This paper summa-
rizes recent studies towards the direct use of inclusion bodies in biotechnol-
ogy and for the detection of bottlenecks in the folding pathways of specific
proteins. We also review the major biophysical methods available for
revealing fine structural details of aggregated proteins and which informa-
tion can be obtained through these techniques.
Abbreviations
DAAO,
D-amino acid oxidase; GFP, green fluorescent protein; IB, inclusion body; TF, trigger factor.
2408 FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS
irreversibly lead to the formation of aggregates (for an
excellent review on protein folding in the cytoplasm
see [6] and references therein).
As translation is a relatively slow process (it can
take up to 75 s to synthesize a protein 300 amino acids
long) and proteins larger than around 100 amino acids
fold slowly [7], cells developed a series of mechanisms
to avoid the exposure of aggregation-prone proteins to
the cytoplasm. As first line of defence, around 40
amino acids of the nascent polypeptide can be accom-
modated inside the ribosome exit tunnel and it has
been demonstrated that secondary (mainly helical)
structure formation is possible inside the tunnel [8].
Outside the ribosome, de novo folding of a growing
chain is facilitated by a number of chaperones: the
trigger factor (TF), the DnaK, DnaJ, GrpE system

evidence for age-dependent protein aggregation also in
bacterial cells [11] and mechanisms to neutralize it
have been characterized in E. coli [12]. If IBs are pres-
ent in a cell, as they tend to aggregate at one extremity
of the bacterium, cell division will produce an IB-free
cell (healthier, young and with higher growth rate) and
an IB-containing one that will grow more slowly [13].
Half of the bacterial progeny will thus have better fit-
ness: ageing is not avoided at single cell but at popula-
tion level.
Fig. 1. Protein biosynthesis and aggregation under normal and
stress conditions. (A) Under normal conditions, nascent polypep-
tides either can fold autonomously or require the help of folding
chaperones. Aberrant protein products due to translation errors and
misfolding are handled by the quality control system, composed of
refolding chaperones and proteases. The system is energetically
demanding (most processes are ATP-dependent) but drives the
equilibrium towards the native, folded state [10]. (B) Under most
stress conditions equilibrium is shifted toward the formation of
aberrant products (red lines). This is naturally counteracted by cellu-
lar optimization strategies already present at the source (DNA, pro-
tein sequences and regulation of expression levels) or induced
upon exposure to stress conditions (upregulation of the quality con-
trol machinery). Heterologous protein overproduction, however, can
further affect this delicate balance by competing for available
resources (ribosomes, chaperones but also ATP).
P. Gatti-Lafranconi et al. Potential of bacterial inclusion bodies
FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS 2409
Rate and ‘quality’ of protein synthesis can favour
misfolding over folding. Intuitively, an increase in the

regions [18] is believed to kinetically promote folding
and the burial of aggregation-prone patches in the
core of the native structure. Although DNA and pro-
tein sequences evolved to optimize translation and
folding efficiency, even a single silent mutation can
induce IB formation, while amino acid replacements
that alter the chemical properties of the polypeptide
will easily result in increased aggregation propensity.
During recombinant protein production, heterologous
proteins will not have their sequence optimized for
expression in E. coli and therefore suffer from poor
folding efficiency even if expression levels are kept
low.
In microbial cell factories, overproduced proteins
can represent up to 90% of the total protein content
and cause the failure of the quality control system that
will result in the accumulation of misfolded proteins
first and eventually lead to the formation of IBs. This
process is highly protein-dependent, driven by DNA
and protein sequences, as discussed above, but can
also be affected by specific folding requirements (i.e.
disulfide bonds) or transcend the folding capability of
E. coli. Other causes of aggregation are heat or oxida-
tive stresses, environmental conditions that cells are
likely to face in natural environments and biotechno-
logical applications. Growth above optimal tempera-
ture eventually results in massive protein unfolding
while reactive oxygen species cause fragmentation and
chemical modification of side chains. Both these events
raise the aggregation propensity of proteins in the

ferent cellular responses depending on their properties,
particularly for what concerns aggregation propensity.
Reports on the upregulation of the quality control sys-
tem upon the accumulation of misfolded proteins in
the cytoplasm suggest that this mechanism shares simi-
lar features with the heat-shock response, which causes
the upregulation of genes controlled by the transcrip-
tion factor r
32
. r
32
regulates the expression of genes
coding for known heat-shock proteins (which include
chaperones and proteases) and its own activity depends
on the same chaperones that it regulates [23,24]. It is
believed that, under non-stress conditions, chaperones
Potential of bacterial inclusion bodies P. Gatti-Lafranconi et al.
2410 FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS
act as anti-sigma factors, inhibiting r
32
activity
through an induced conformational change [24,25].
When the number of misfolded proteins increases in
the cell, chaperones are saturated and the equilibrium
shifts toward the free version of r
32
, leading to induc-
tion of the stress response.
Nevertheless, the nature and variability of the
recombinant protein stress response suggests a

that membrane lipids may act as a second stress sensor
responsive to the aggregation state of the recombinant
protein [33,34].
The bright side of IBs: from
recombinant protein reservoir to tools
for basic investigation and direct
application in biotechnology
Before the last decade, the properties of protein aggre-
gates knew little glory while most studies pursued
either solubility improvement or denaturation ⁄ renatur-
ation of purified IBs. Within the first line, the most
successful techniques are fusion with solubility tags,
use of molecular and chemical chaperones and modu-
lation of the expression conditions to reduce the rate
of protein biosynthesis [9,35–37], whereas in the second
major efforts are devoted to optimizing the refolding
process so as to regain highest biological activity
(reviewed in [38]). Only during the last decade has a
deeper knowledge of the structural and functional
properties of IBs drawn researchers’ attention to the
possibility to control the conformation of aggregated
proteins, paving the way for the use of IBs in a series
of studies and applications that were difficult to envis-
age only a few years ago.
Such developments require that IBs can be charac-
terized in fine detail, their structure and aggregation
process monitored and controlled. Having structural
information in hand would enable these methods to be
applied in an informed fashion and thus allow a fine
modulation of the aggregation process. In the next sec-

form at early stages of the process while, at later times,
these assemblies merge into one or two large aggre-
gates localized at the poles of the cells [39,40]. In vivo
aggregation can also be monitored in real time label-
ling the target protein with the tetra-Cys sequence tag
(Cys-Cys-X-X-Cys-Cys) that specifically binds a fluo-
rescein analogue containing two arsenoxides (FIAsH).
In this approach, the tetra-Cys motif is introduced by
mutagenesis into the protein sequence at a specific
position where its accessibility and binding to FIAsH
will depend on the folding state of the protein. In this
way, FIAsH fluorescence reports on protein stability
and aggregation within cells [41]. Other applications of
fluorescence-based analysis rely on proteins within IBs
retaining native-like structure and activity. For exam-
ple, it was shown that in IBs formed by a GFP-fusion
protein fluorescence emission was higher in the core of
the aggregates than in their external shell [42]. This
observation ruled out the possibility that the biological
activity retained by IBs depends on native-like proteins
passively trapped in the aggregate and instead attrib-
uted this distribution to the specific mechanisms of
protein deposition and removal, and further suggested
that aggregated proteins can complete their folding
and activation process once deposited in IBs [42]. Pro-
tein–protein interactions within IBs have also been
studied using higher resolution fluorescence approaches
such as the Fo
¨
rster resonance energy transfer (FRET)

Furthermore, both electron microscopy and, in partic-
ular, atomic force microscopy image the surface mor-
phology of the sample at nanometric resolution [51]
and allowed amyloid-like fibrils to be detected in
freshly purified IBs of the human bone morphogenetic
protein-2 (fragment 13–74) [52] and of the prion of the
filamentous fungus Podospora anserine HET-s (frag-
ment 218–289) [53]. Fibrillar structures became more
evident after IB incubation at 37 °C for 12 h [52] or in
the presence of proteinase K [44,54].
The structural properties of IBs at molecular level
have been investigated at a resolution ranging from
protein backbone conformations to single residues by
several optical spectroscopies, such as FTIR, Raman,
Fig. 3. Transmission electron micrograph of IBs within E. coli cells.
The picture shows IBs formed by GFP fused to an aggregation-
prone domain and the immunolocalization of GFP. Courtesy of Ele-
na Garcı
´
a-Fruito
´
s and Antonio Villaverde.
Potential of bacterial inclusion bodies P. Gatti-Lafranconi et al.
2412 FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS
CD and fluorescence, as well as by NMR and X-ray
diffraction.
FTIR spectroscopy allows the study of protein sec-
ondary structures and aggregation through the analysis
of the amide I band, occurring in the 1700–1600 cm
)1

)1
have been used to follow the
kinetics of IB formation within a growing culture of
E. coli. To exemplify this approach, Fig. 4A reports
the second derivative spectrum of E. coli cells during
production of a recombinant lipase. Six hours after
induction at 37 °C the protein is mainly deposited in
aggregates, as can easily be determined based on the
appearance of a shoulder at  1627 cm
)1
that has no
counterpart in the control cells and is attributed to
intermolecular b-sheet structures in protein aggregates.
Subtraction of the spectrum of control cells allowed
the spectral component (1627 cm
)1
) unique to aggre-
gates to be resolved in more detail (Fig. 4B) and the
kinetics of IB formation at different temperatures,
namely at 37 and 27 °C, the latter compatible with the
partitioning of the recombinant protein between solu-
ble and insoluble proteins, to be monitored and com-
pared [57]. Spectra of IBs (Fig. 4C) purified from cells
revealed that the intermolecular b-sheet component of
protein aggregates, peaked at 1627 cm
)1
, was higher at
the higher temperature, while proteins embedded in
IBs formed at 27 °C retained more native-like a-helical
content (1656 cm

)1
due to intermolecular b-sheets in
aggregates is well resolved allowing the kinetics of IB formation
within intact cells to be monitored. The same analysis performed at
27 °C is shown (dotted-dashed line). (C) Second derivative absorp-
tion spectra of IBs extracted after 10 h from induction at 27 °C
(dotted-dashed line) and 37 °C (continuous line).
P. Gatti-Lafranconi et al. Potential of bacterial inclusion bodies
FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS 2413
unexplored in IB studies, since relevant information on
disulfide bond formation and on solvent accessibility
of specific amino acid side chains can be obtained [63].
The presence of b-sheet structures in extracted IBs
can also be detected by far UV CD [52,54], even if it is
not easy to discriminate between intramolecular and
intermolecular b-sheets. The use of this spectroscopic
technique for the study of IB aggregates is often
limited by the intrinsic insolubility of the samples,
responsible for a high level of light scattering distur-
bances and signal loss.
The characteristic presence of b-sheet structures
within extracted IBs has also been confirmed by X-ray
diffraction. Spectra typically display two circular
reflections around 4.7 A
˚
and 10.2 A
˚
, respectively,
assigned to the spacing between strands within a
b-sheet and between b-sheets. The circular shape of

obtained also by other spectroscopic techniques such
as CD and X-ray diffraction [52]. It is noteworthy that
NMR-based approaches, such as solid-state NMR
13
C–
13
C proton-driven spin diffusion and liquid-state
NMR H ⁄ D exchange experiments, offer the unique
possibility of comparing at the residue-specific level
protein aggregates of different types, such as IBs, amy-
loid fibrils and thermal aggregates [53,64]. The out-
comes of these NMR experiments could therefore
allow the aggregate residue-specific structural proper-
ties to be correlated with their functional features, such
as enzymatic activity or cellular toxicity.
Exploitation of IBs in biotechnology
and in protein science
It is widely recognized that proteins can aggregate in
IBs in different folding states that can eventually coex-
ist within the same aggregates. The conformation
acquired within aggregates is dependent on the nature
of the protein itself [66] but can also be controlled
through the genetic background of the host cells
and ⁄ or manipulation of the experimental conditions.
This novel and in a way revolutionary knowledge has
important consequences in the rationale of handling
and studying IBs. The development of methods to con-
trol and monitor the process of aggregation allows for
the production of aggregated proteins endowed with
residual structure and biological activity that can find

b-galactosidase [67], endoglucanase [68], GFP [69],
a bacterial lipase [57], oxidases [70], kinases [71]
phosphorylases [72] aldolases [73], transglutaminases
[74] and the colony stimulating factor [75]. This knowl-
edge soon generated the idea of directly using IBs in
biocatalysis, thus avoiding the cumbersome step of
resolubilization. Since recovery of IBs from cell
extracts can be quite easily achieved, this method
could be of broad scope, provided aggregated proteins
retain enough biological activity. Unfortunately, so far
the comparison of the specific activity of soluble and
Potential of bacterial inclusion bodies P. Gatti-Lafranconi et al.
2414 FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS
aggregated proteins has been performed only sporadi-
cally although the competitiveness of IB catalysis
depends on the balance between a possible reduction
of specific activity and the advantages produced by
avoiding solubilization steps. Data available show that
depending on the protein and the production protocol
the biological activity of aggregates can vary from 11%
[69] to nearly 100% [68] of the soluble counterpart.
IBs embedding native-like proteins are also proposed
as a source of pure recombinant proteins that can be
easily released upon mild treatments that avoid chemi-
cal disruption of cells and denaturation of the aggre-
gates. Protein–protein interactions are in fact weaker
and ‘relaxed’ IBs can be dissolved in mild detergent at
low concentration. Since proteins have not been dena-
tured during solubilization, there is no need to intro-
duce refolding steps, which is of great advantage since

In the same conceptual frame – making soluble pro-
teins insoluble – other authors have developed a self-
assembly complex in which IBs are formed through
in vivo aggregation of polyhydroxybutyrate synthase
PhaC carrying at its N-terminus a negatively charged
coil [78]. Aggregates of this protein expose on their
surface charged regions that can bind active soluble
enzymes tagged at their C-terminus with a positively
charged coil.
In both cases, examples available are still too few to
be generalized in a broad scope experimental
approach. However, the importance of IBs as direct or
indirect immobilization carriers might increase when,
for instance, different enzymes ⁄ proteins can participate
in the same aggregate to build a multifunctional aggre-
gated catalyst.
Finally, but not less important, it should be con-
sidered that pathways of protein folding are reflected
in the formation of IBs and in their structure. Study-
ing protein aggregates can therefore provide a first
glimpse about the occurrence of folding-limiting
steps. The finding that aggregates of several different
proteins, for example INF-a-2b [56], a bacterial
lipase [57], a mutant of the Ab42 Alzheimer peptide
[79] and GFP [69] can be endowed with substantial
amounts of native structure led to the conclusion
that the process of intracellular aggregation can
involve proteins in a continuum of conformational
states. This idea is well substantiated by the demon-
stration that different conformations of the same

FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS 2415
IBs produced in different conditions can be considered
as an easy tool to detect the presence of critical
folding intermediates to be characterized with other
techniques.
To conclude, we believe that a truly successful
understanding and exploitation of IBs requires an
advanced understanding of cellular and protein mech-
anisms leading to aggregation as well as powerful
biophysical detection methods. Reported examples
highlight the potential of these approaches in creating
new generation protein depositories and biocatalysts.
Acknowledgements
S. M. D. and M. L. acknowledge support by FAR
(Fondo di Ateneo per la Ricerca) of the University of
Milano-Bicocca. P. G. -L. is the recipient of a Marie
Curie Intra-European F ellowship. A. N. and D. A. a ckno-
wledge postdoctoral fellowships of the University of
Milano-Bicocca.
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