Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology Pietro Gatti-Lafranconi1,*, Antonino Natalello2,*, Diletta Ami2, Silvia Mar
Trang 1Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology
Pietro Gatti-Lafranconi1,*, Antonino Natalello2,*, Diletta Ami2, Silvia Maria Doglia2and Marina Lotti2
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 these conditions, folding becomes a kinetic race against aggregation: although the native state is thermodynam-ically favoured [5], aggregation can trap folding inter-mediates into non-native folding landscapes that,
in the absence of further control mechanisms, would
Keywords
aggregation; amyloid-like structures;
biocatalysis; electron and optical
microscopies; fourier transform infrared
spectroscopy; inclusion bodies; IB structural
properties; native-like conformation;
recombinant proteins; stress response
Correspondence
S M Doglia, M Lotti, Department of
Biotechnology and Biosciences, State
University of Milano-Bicocca, Piazza della
Scienza 2, 20126 Milano, Italy
Fax: +39 02 64483565
Tel: +39 02 64483459
E-mail: silviamaria.doglia@unimib.it;
marina.lotti@unimib.it
*These authors contributed equally to this
work
(Received 28 January 2011, revised 20
March 2011, accepted 5 April 2011)
doi:10.1111/j.1742-4658.2011.08163.x
Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation However, several reasons can cause the failure
of such defences, among them mutations, stress conditions and high rates
of protein synthesis, all common consequences of heterologous protein 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.
Trang 2irreversibly 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
and the GroEL–GroES pair A comprehensive review
of the folding process transcends the aim of this paper
and can be found in [9,10] and references therein The
folding machinery allows most proteins to efficiently
reach their native state but, even in non-stress
condi-tions, some molecules fail to do so When the folding
machinery fails, cells deal with unfolded proteins
through alternative mechanisms Holding chaperones
(IbpA⁄ B, Hsp31 and Hsp33) temporarily bind
misfold-ed peptides on their surfaces and present them to
DnaK⁄ J or GroEL ⁄ ES AAA+ proteases act on
formed aggregates triggering the degradation of
mis-folded proteins while ClpB releases them from
inclu-sion bodies (IBs) and presents unfolded polypeptides
to the (re)folding machinery Altogether, under
physio-logical conditions, this quality control system can
sense, react to, control and reduce to negligible levels
the amount of partially unfolded and aggregated
pro-teins in the E coli cytoplasm (Fig 1A)
Stress conditions, however, cause the impairment of
the cellular quality control system, thus inducing
mis-folded proteins to accumulate in the cytoplasm as
insoluble aggregates, or IBs (Fig 1B) In the case of
E coliand other bacteria used as microbial cell
facto-ries, main stress conditions are ageing, rate of protein
synthesis, mutations and aberrant protein biogenesis,
environmental (usually heat or oxidative) stress and
heterologous protein production
Ageing is mostly known to induce protein
aggrega-tion-related diseases in higher eukaryotes but there is
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).
Trang 3Rate and ‘quality’ of protein synthesis can favour
misfolding over folding Intuitively, an increase in the
concentration of nascent polypeptides makes the
fold-ing process more severe and this is indeed naturally
counteracted by the increase in chaperone
concentra-tion in exponentially growing cells [2] Rate can,
how-ever, be increased above tolerable limits by mutations
and overexpression, as discussed in the next
para-graphs Also, ‘quality’ of protein synthesis is affected
by environmental stress due to an increase in the rate
of translational errors, amino acid misincorporation,
premature chain truncation and incomplete
modifica-tions Such aberrant molecules accumulate in the
cyto-plasm and increase protein aggregation [14]
In general, mutations are retained only if folding
propensity remains above a critical point,
indepen-dently of the advantage that they would provide to
the host [15] Mutations can affect aggregation,
how-ever, even if protein activity is not compromised, even
if no amino acid replacement is introduced: the DNA
sequence itself determines the rate of synthesis There
is indeed evidence for genomic-level optimization of
protein folding at both DNA and protein sequence
levels The distribution of codons in mRNAs has been
found to be unbalanced: as the first 30–50 codons
have low translation efficiency, translation has a slow
start, reducing ribosome clashing, translation stalling
and eventually favouring folding [16] At protein level,
regions in the primary sequence that are intrinsically
aggregation-prone correlate with those having low
folding propensity (and with chaperone dependence)
[17] The localization of ‘fast’ codons around those
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 cytoplasm, either by increasing hydrophobic patch exposure or by altering protein chemical properties that can result in crosslinking and misfolding
Recombinant protein production might induce aggregation and elicit stress responses
Heterologous protein production is by itself cause of toxicity for cells, independently of the nature of the recombinant protein Energy depletion is the most immediate result and is due to both the overproduced protein and the upregulation of those involved in stress responses If degradation of the heterologous protein occurs, even higher energy consumption will result in little product accumulation at the expenses of biomass and growth rate Also, aminoacilated-tRNA depletion triggers the stringent response [19,20] that causes the downregulation of the protein and amino acid biosyn-thesis machinery In a condition of limited resources for protein biosynthesis, competition is won by the recombinant mRNA, causing a decrease in housekeep-ing mechanisms (i.e DNA and protein synthesis), rear-rangements in cellular catabolic rates and slower, if any, growth rate [21] (Fig 1B) The DNA damage-induced SOS response is also reported to be activated and, although there is no agreement about how protein overproduction triggers this response, it is likely that elevated transcription rates of plasmid-encoded genes causes DNA suffering in cells [22]
While these effects occur ubiquitously, overproduced proteins have been reported to specifically trigger dif-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 r32 r32 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
Trang 4act as anti-sigma factors, inhibiting r32 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 r32, leading to
induc-tion of the stress response
Nevertheless, the nature and variability of the
recombinant protein stress response suggests a
far more complex and adjustable ‘heat-shock-like’
mechanism [26] The normal heat-shock response is
transient, fading away shortly after cells are released
from stress, but increased synthesis rates of DnaK,
GroEL chaperones and Lon (the main heat-shock
pro-tease) have been found to last for the whole length of
overproduction The extent and kinetics of the
heat-shock-like response vary among different production
systems and are influenced by the nature of the protein
synthesized: while energy metabolism, SOS response,
nutrient uptake and the core of the heat-shock
response undergo comparable changes, different
recombinant proteins have distinct impacts on
intracel-lular stress control and growth rates [27–29] The small
heat-shock proteins IbpA and IbpB, for example, are
upregulated exclusively when proteins accumulate as
IBs, inhibit IB degradation and reduce the stress
response, thus favouring growth [30,31] A membrane
and a membrane-bound recombinant protein have
opposite effects on growth rate but activate the same
stress-response pattern both at cytoplasm and envelope
level [32] Conversely, in the cytoplasm recombinant
proteins with different aggregation profiles increase the
abundance of the same set of envelope proteins while
membrane composition and permeability specifically
react to the aggregation state of the recombinant
pro-tein It has been suggested that the cell membrane
might react with exquisite sensitivity not only to
aggre-gation but even to the complexity of the aggregates
(whether soluble aggregates or large insoluble IBs) and
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-tion we describe and illustrate with some examples the major tools available for the structural analysis of pro-teins within aggregates and of aggregates within cells Synergic to the latter goal are computational methods allowing the identification of aggregation-prone regions within protein primary sequences (reviewed by Hamodrakas in this issue)
Fig 2 Methods for the characterization of IBs (A) Scheme of IB formation and structural properties Folding intermediates form sol-uble aggregates that merge in one or two IBs per cell The polypep-tides embedded in IBs can retain native-like structure and activity Moreover, IBs can acquire amyloid-like features Possible applica-tions related to the peculiar IB structural properties are indicated (B) Principal methods of investigation of IB formation and character-ization.
Trang 5Structural properties of IBs: a review
of the methods
We provide in the following an updated view about
the principal biophysical methods available for the
characterization of proteins aggregated in IBs and
summarize the information generated by their
applica-tion (Fig 2)
The aggregation of recombinant proteins can be
monitored in vivo by fluorescence spectroscopy and
microscopy if the target protein is fused to a
fluores-cent partner such as the green fluoresfluores-cent protein
(GFP) or its variants [39] Using this approach it was
determined that multiple, small and soluble aggregates
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)
in which interacting proteins are labelled by two
differ-ent fluorescdiffer-ent probes [43] Higher FRET efficiency
was obtained when the two probes were fused to the
same peptide rather than to different ones, suggesting
that the process of aggregation is highly
protein-spe-cific [44] The spatial resolution of optical
microscop-ies, including fluorescence microscopy, is of the order
of 0.1 lm (in the image X, Y plane) due to the
diffrac-tion limit of the employed light Even in laser scanning
confocal microscopy, the highest resolution of about
0.5 lm is obtained in the Z direction [45]
Electron and atomic force microscopies reach a na-nometric – and even subnana-nometric – resolution but they rely on a more invasive approach to the sample
In transmission electron microscopy, thin sections of fixed cells show IBs as spherical or ellipsoidal electron dense structures [46,47] and purified IBs appear as spherical, ellipsoidal or cylindrical particles of 0.5– 1.8 lm characterized by a smooth and porous surface
in both scanning and transmission electron microscopy (Fig 3) [46,48] The porous structure of IBs, also con-firmed by sedimentation techniques [49], is of relevance
in view of a direct application of active aggregates in biocatalysis: thanks to the porous and hydrated IB structure, substrates and products can diffuse inside and outside making IBs useful depositories of highly purified enzymes Electron microscopy was also applied to studying the shape and surface to volume ratio of protein aggregates used as biomaterials in applications where these features are of relevance [50] 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 37C 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.
Trang 6CD 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
absorption region, which is due to the CO stretching
vibration of the peptide bond (reviewed in [55] and
ref-erences therein) Absorption of the different secondary
structures of the proteins overlaps in this spectral
range and can be resolved by resolution enhancement
approaches, such as the second derivative analysis of
the spectra In this way, the secondary structure
com-ponents appear as negative peaks in the derivative
spectrum and each peak can be assigned according to
its wavenumber For instance, in water a-helices and
random coils absorb between 1660 and 1648 cm)1,
intramolecular b-sheets between 1640 and 1623 cm)1
and around 1686 cm)1, whereas intermolecular b-sheet
absorption in protein aggregates is found between
1630 and 1620 cm)1 and around 1695 cm)1 FTIR
(micro)spectroscopy allows protein secondary
struc-tures and aggregation to be studied also within
com-plex biological systems, i.e whole intact cells [56–58],
tissues [59] and whole organisms [60] Moreover,
changes in the intensity of the aggregate spectral
com-ponent around 1625 cm)1have 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 37C 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 27C, the latter compatible with the
partitioning of the recombinant protein between
solu-ble and insolusolu-ble 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 27C retained more native-like a-helical
content (1656 cm)1) These results suggest FTIR
(micro)spectroscopy as a technique of choice also in
the study of the influence of the physiology of
expres-sion (i.e temperature, induction, formation of disulfide
bonds) on the kinetics of aggregation and on the
struc-ture of aggregated proteins [57,61]
Another vibrational technique that can be employed
to characterize the structural properties of IBs is Raman (micro)spectroscopy, where the inelastic scat-tering of laser light from the sample is detected Pio-neering work of Przybycien et al detected in IBs formed by recombinant b-lactamase an increased level
of b-sheet structures and the retention of native-like a-helix content [62] This technique can be considered complementary to FTIR spectroscopy, since the two methods detect different vibrational modes of the sam-ple Raman spectroscopy is more sensitive to the amino acid side chain response [63] while – as dis-cussed above – FTIR is more sensitive to the backbone amide I vibrations We believe that Raman (micro)spectroscopy could offer advantages still
Fig 4 FTIR analysis of the aggregation of a recombinant protein in
E coli (A) Second derivatives of the FTIR absorption spectra of
E coli cells synthesizing a recombinant lipase from Pseudomonas fragi (PFL) at 37 C after 6 h from induction (continuous line) and of the control cells (dashed line) (B) Second derivative of the differ-ence spectrum between cells producing the recombinant protein and control cells reported in (A) (continuous line) In this subtracted spectrum, the band at 1627 cm)1due 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 inducabsorp-tion at 27 C (dotted-dashed line) and 37 C (continuous line).
Trang 7unexplored 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
these reflections has been suggested to arise from not
strongly aligned b-sheets within IBs [52,64]
NMR spectroscopy has been widely applied in
pro-tein science, since it enables detailed structural
infor-mation at the specific residue level up to the
three-dimensional structure of the protein to be obtained In
particular, solid state NMR rotational-echo
double-resonance (REDOR) has been applied to IBs, both
extracted and within intact cells [65] In this approach,
the backbone carbonyl and nitrogen are labelled (13CO
and15N) for each amino acid, since its13CO chemical
shift allows information to be obtained on local
con-formation In this way, Curtis-Fiske et al were able to
identify native a-helices of the N-terminal 185 residues
of the functional domain of the HA2 subunit of the
influenza virus hemagglutinin protein and to detect
conformational heterogeneity of the protein within IBs
[65] NMR spectroscopy has also been applied to
localize b-sheet structures in protein aggregates, mainly
by hydrogen⁄ deuterium (H ⁄ D) exchange experiments
that allow residue-specific backbone amides protected
from solvent exchange because they are involved in
hydrogen bonds to be detected The assignment of
sol-vent-protected residues to b-sheet structures can be
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
13C–13C 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 direct use in biotechnology In addition, a detailed analysis of the mode of building and of the structure
of aggregates can be useful to dissect pathways and bottlenecks in the folding of specific proteins, for example those containing disulfide bonds or requiring cofactors, multidomain proteins, fusion proteins In the following we summarize recent progress in this field, whereas the use of IBs in the study of amyloid aggregation is developed in the accompanying review paper by Garcı´a-Fruito´s and colleagues
Two very relevant accomplishments towards IB exploitation in biotechnology are based on the ability
to enrich aggregates in native-like structured proteins making them suitable for direct use in biocatalysis and⁄ or as a source of relatively pure proteins that can
be released through mild solubilization Given that aggregation often cannot be fully avoided – or is even considered an advantage – the same experimental
‘tricks’ developed to improve the solubility of recombi-nant proteins (reviewed in [35]) can be applied to pro-duce IBs mostly composed of native-like, although not soluble, recombinant proteins
The list of recombinant proteins that precipitate in IBs in a conformation permissive for biological activity has progressively grown since researchers started to measure this parameter and includes, among others, 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
Trang 8aggregated 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
solubilization⁄ refolding is often a critical step in the
production of recombinant proteins Interestingly the
approach has been successfully tested with proteins not
related in their structure, among them the granulocyte
colony stimulating factor, GFP and a truncated form
of the tumour necrosis factor [76]
An innovative evolution towards IB-based catalysis
exploits the idea of forcing otherwise soluble proteins
to aggregate in IBs This method is proposed as an
alternative to the better known procedures of enzyme
insolubilization via immobilization on carriers or via
aggregation by crosslinking (reviewed in [77]) The
pro-tein of interest is fused to an aggregation-prone moiety
promoting the aggregation of the chimeric polypeptide
The cellulose-binding module, a very poorly soluble
protein, was used to induce intracellular deposition of
the recombinant d-amino acid oxidase (DAAO) from
Trigonopsis variabilis, an enzyme used in the synthesis
of 7-amino cephalosporanic acid [70] The observation
that DAAO IBs retained specific activity close to that
of the soluble enzyme and were resistant under
condi-tions that inactivate free DAAO substantiated the
fea-sibility of this approach, which was then applied also
to a maltodextrin phosphorylase [72], a polyphosphate
kinase [71] and a sialic acid aldolase [73] Clearly,
fusion with the cellulose binding domain did not
inter-fere with the correct folding of the partner protein that
aggregated in a form endowed with biological activity
(in the case of DAAO this means also ability to bind
the cofactor)
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 polypeptide coexist in IBs [80] However, the struc-ture of aggregated TEM1-b-lactamase inside IBs could not be affected by any of the usual means [81] In this particular case it was therefore con-cluded that TEM aggregation is only controlled by the amino acid sequence and not by the kinetics of folding, since changing the rate of biosynthesis did not result in structural changes in the aggregates This result was interpreted as evidence about the existence of a single specific folding step critical for the protein undergoing either aggregation or native folding
Analysis of the modulation of aggregation in bacte-ria was also of support in clarifying critical steps of oxidative folding of bovine b-lactoglobulin b-Lacto-globulin carries five cysteine residues, four of which link in disulfide bridges, raising questions about the role (if any) of the free thiol during in vivo folding Upon overproduction in E coli cells optimized for the intracellular formation of disulfide bonds, it was observed that a mutant protein deprived of the unpaired Cys was more prone to aggregation than the wild type, pointing to a contribution of the free thiol
in the pathway leading to the formation of native bonds [61]
The number of proteins studied up to now is still too limited to try to generalize which structural, sequence and kinetic properties might dictate the fine detail of aggregation However, structural analysis of
Trang 9IBs 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 Fellowship A N and D A
ackno-wledge postdoctoral fellowships of the University of
Milano-Bicocca
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