Journal of Biotechnology 125 (2006) 110–113

Short communication

Protein activity in bacterial inclusion bodies correlates with

predicted aggregation rates
Natalia Sánchez de Groot, Salvador Ventura ∗
Departament de Bioquı́mica i Biologia Molecular, Institut de Biotecnologia i de Biomedicina,
Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain

Received 14 December 2005; received in revised form 8 February 2006; accepted 14 February 2006

Abstract

Recent data show that protein aggregation as bacterial inclusion bodies does not necessarily imply loss of biological activity.
Here, we investigate the effect of a large set of single-point mutants of an aggregation-prone protein on its specific activity once
deposited in inclusion bodies. The activity of such aggregates significantly correlates with the predicted aggregation rates for
each mutant, suggesting that rationally tuning the kinetic competition between folding and aggregation might result in highly
active, inclusion bodies. The exploration of this technology during recombinant protein production would have a significant
biotechnological value.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Inclusion bodies; Recombinant protein expression; Protein aggregation; Protein folding; Escherichia coli

Protein misfolding is a common event during bacte-
rial over-expression of recombinant genes (Baneyx and
Mujacic, 2004). The aggregation of insoluble polypep-
tide chains as inclusion bodies (IBs) is the main bottle-
neck in protein production, narrowing the spectrum of
relevant polypeptides obtained by recombinant tech-
niques and hampering the development of top pri-
ority research areas such as the de novo design of
novel proteins, the rational modification of natural pro-
teins and structural genomics and proteomics. Being
∗ Corresponding author. Tel.: +34 93581 4147;
fax: +34 93581 1264.
E-mail address: salvador.ventura@uab.es (S. Ventura).
widespreadly believed that IB proteins are biologically
inert and therefore useless in bioprocesses, many bio-
logically relevant proteins have been disregarded for
commercialisation.
We have shown recently that not only the aggre-
gation of different recombinant proteins as bacterial
IBs does not necessarily inactivate them but also that
active IBs can be used in suspension as efficient cat-
alysts for bioprocesses (Garcia-Fruitos et al., 2005).
In concrete, the over-expression of a fusion of the
aggregation-prone, Alzheimer-related peptide A␤42 to
green fluorescent protein (GFP) resulted in highly fluo-
rescent IBs. In the present study, we have quantitatively
investigated the biological activity of the IBs formed

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.02.026
 N.S. de Groot, S. Ventura / Journal of Biotechnology 125 (2006) 110–113 111

Fig. 1. Fluorescence emission of IBs formed by A␤42-GFP variants (a) Fluorescence microscopy images of cells expressing wild type (A)
and F19D (B) A␤42-GFP. Cells were imaged at 40-fold magnification under UV light. Site-directed mutagenesis was performed using the
QuickChange kit from Stratagene. All constructs were verified by DNA sequencing. Culture and protein expression conditions were as previously
described (Garcia-Fruitos et al., 2005) with the exception that cultures were collected 8 h after induction to ensure equilibrium. (b) Specific
activity of different A␤42-GFP variants. The data are ordered by decreasing specific fluorescence at 510 nm (fluorescence U/␮g protein).
Inclusion bodies were purified by a detergent washing protocol as described (Carrio et al., 2000) and used in suspension for activity analysis.
The GFP fluorescence of a 1 ml of suspension was recorded at 510 nm, using an excitation wavelength of 450 nm (emission and excitation slits
widths 5 mm). Data were corrected for buffer signals. At least three different scans were averaged for each protein sample. For the determination
of inclusion body protein, these structures were resuspended in denaturing buffer (Laemmli, 1970). After boiling for 20 min, appropriate sample
volumes were loaded onto denaturing gels. Gels were scanned at high resolution and bands quantified by using the Quantity One software from
Bio-Rad, by using appropriate protein dilutions of known concentration as controls. Determinations were always done within the linear range
and they were used to calculate the specific activity values.
112 N.S. de Groot, S. Ventura / Journal of Biotechnology 125 (2006) 110–113
by 20 different mutants of the A␤42-GFP fusion pro-
tein, to understand the rules underlying protein deposi-
tion during recombinant protein expression but also to
explore the possibility of, through protein engineering,
deliberately obtaining highly active proteins, useful for
bioprocesses in IB form.
We used a set of 20 A␤42-GFP fusion variants
differing only in the residue at position 19 to eluci-
date if the primary sequence of a polypeptide might
influence the occurrence of active protein in IBs
when over-expressed in Escherichia coli. In wild type
A␤42 peptide the residue 19 is a Phe; each of the
19 mutants analyzed in this study possess a differ-
ent natural amino acid in this position. Position 19
has been shown to strongly affect the aggregation of
this Alzheimer-related peptide (Wood et al., 1995)
thus being, a priori, a good target to test the effect of
sequence changes on the specific activity of aggregated
protein.
Upon overproduction, all 20 proteins formed fluo-
rescent cytoplasmic inclusion bodies in E. coli. The
different IBs were purified from the insoluble cell frac-
tion and the intensity of the green fluorescence emitted
by GFP embedded in IBs measured. The fluorescence
emission changed from variant to variant (Fig. 1A). The
specific fluorescence of the inclusion bodies formed
by the most fluorescent mutant (F19D) was fourfold
higher than those exhibited by the wild type aggregated
protein (Fig. 1B).
In order to rationalize how sequence variation pro-
motes changes in IBs activity we studied the corre-
lation between IBs specific fluorescence and the pre-
dicted aggregation rates for the different A␤42-GFP
variants according to Eq. (1) (Fig. 2). A highly sig-
nificant inverse correlation was observed (r = 0.941,
p = < 0.0001), strongly suggesting that the final amount
of active protein in a given IB depends on how fast the
aggregation event occurs.
The results in this report indicate that the accu-
mulation of active protein in IBs is not anecdotic
but that it could be a general feature in recombinant
protein production. More interestingly, we show that
the aggregation of protein as IBs during recombi-
nant protein expression is not an unspecific and pas-
sive process but rather a kinetically controlled event
which speed depends specifically on the polypeptide
nature and probably mainly on the sequence of certain
aggregation-prone regions.
Fig. 2. Correlation between IBs specific fluorescence and pre-
dicted aggregation propensities of A␤42-GFP fusions. Specific
fluorescence of A␤42-GFP fusions IBs were plotted vs. the
changes in aggregation rates predicted by the Eq. (1) (devel-
oped by Chiti et al. (2003)). This approach assumes that ␤-sheet
propensity, hydrophobicity and charge are independent factors,
which affect the aggregation of a protein, in an additive manner.

v 
mut
ln = A
hydrophobicity + B
␤-sheet propensity
vwt
+ C
charge (1)
where vmut and vwt correspond to the predicted aggregation rates
of the mutant and wild type sequences, respectively and
charge is
the difference in the net charge of the polypeptide introduced by the
mutation. A, B and C values are constants determined experimentally
from the analysis of a large set of mutants of Acylphosphatase.
It is assumed, but scarcely proven in vivo, that aggre-
gation competes with folding (Smith and Hall, 2001).
The results herein constitute one of a few direct evi-
dences of this theory. Fluorescence is indicative of
both correct GFP folding and chromophore formation.
Accordingly, the observed differences indicate differ-
ent amount of active GFP trapped in the aggregates.
Assuming that, in order to attain functionality, the
attainment of a GFP native structure should precede
aggregation, and that the time needed for this process
is identical for GFP in all fusions assayed, then IBs flu-
orescence emission relates to the time the A␤42-GFP
variant was soluble after its synthesis and before to
its aggregation. Thus, fluorescence probably inversely
reflects the in vivo aggregation rate as suggested by the
highly significant correlation found between IBs flu-
orescence and predicted aggregation rates. The faster
the fusion protein aggregates, the lower its fluorescence
emission and vice versa, in such a way that fluores-
 N.S. de Groot, S. Ventura / Journal of Biotechnology 125 (2006) 110–113
cent molecules in IBs are those whose aggregation was
slow enough to permit prior GFP folding. It is impor-
tant to note that usually the productive folding of the
GFP reporter has been directly related to the solubil-
ity of the upstream fused protein when over-expressed
in E. coli (Waldo et al., 1999). According to our data
it could have eventually been indicative of the fold-
ing performance of the fused protein rather than of its
solubility–insolubility.
Overall, this system appears as a valid one to explore
in vivo the kinetic competition between folding and
aggregation events. As shown here, tuning aggrega-
tion speed might result in highly active (fluorescent)
IBs, which being highly pure, compact but porous
and hydrated protein microparticles might be used as
bio catalysers opening intriguing possibilities for the
biotechnological industry.
Acknowledgments
We thank Prof. Antonio Villaverde for manuscript
revision and suggestions. We also thank Prof. Josep
Vendrell and Prof. Francesc Xavier Aviles for lab
facilities. S.V. is recipient of a “Ramón y Cajal” con-
tract awarded by the MCYT-Spain and co-financed
113
by the Universitat Autònoma de Barcelona (UAB,
http://www.uab.es/), and founded by PNL2004-40
(UAB) and 2005SGR-00037(AGAUR).
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