Bioengineered Bugs 2:4, 222-225; July/August 2011; © 2011 Landes Bioscience
How to break recombinant bacteria
Does it matter?
Escarlata Rodríguez-Carmona, Antonio Villaverde and Elena García-Fruitós*
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia; Universitat Autònoma de Barcelona; CIBER en Bioingeniería;
Biomateriales y Nanomedicina (CIBER-BBN); Bellaterra, Barcelona Spain
R
ecombinant proteins and other
materials of industrial interest produced in Escherichia coli are usually
retained within the bacterial cell, in the
cytoplasmic space, where they have been
produced. Different protocols for cell
disruption have been implemented as an
initial downstream step, which keeps the
biological and mechanical properties of
the process products. Being necessarily
mild, ©2
these
approaches
often
result
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95–99% cell disruption, what is more
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than acceptable from the yield point of
view. However, when the bacterial product are nano or microparticulate entities
that tend to co-sediment with entire bacterial cells, the remaining undisrupted
bacteria appear as abounding contaminants, making the product not suitable
for a spectrum of biomedical applications. Since bacterial inclusion bodies are
now seen as bacterial materials valuable
in different fields, we have developed an
alternative cell disruption protocol that
permits obtaining fully bacterial free
protein particles, keeping the conformational status of the embedded proteins
and the mechanical properties of the full
aggregates.
Key words: cell disruption, recombinant
products, bacterial cytoplasm, isolation
protocol, biomaterials, downstream step
Submitted: 03/11/11
Revised: 04/15/11
Accepted: 04/18/11
DOI: 10.4161/bbug.2.4.15778
*Correspondence to: Elena García-Fruitós;
Email: [email protected]
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Escherichia coli is still the most commonly
used microorganism for the production of
recombinant proteins, specially for analytical purposes but also for biotechnological and pharmaceutical industries.1 Being
a Gram negative bacteria, E. coli usually mostly retains recombinant proteins
within the cytoplasm unless a specific
leader peptide is joined to the target protein. However this strategy is not always
Bioengineered Bugs
successful. E. coli is also quickly becoming the main bacterial host for the production of a wide range of biomaterials and
nanoparticulate entities,2,3 such as recombinant production of some natural and
new unnatural polymers (polyhydroxyalkanoates (PHA),4 and lactate-based
polyesters,5 among others), phages,6 virus
like particles,7 flagella and flagella components,8 metal and magnetic particles9
and inclusion bodies (IBs).10 Many of
these materials have showed important
demonstrable values in conventional and
innovative medicines,11 by providing
unusually tuneable properties of interest over those exhibited by substances or
particles obtained by chemical synthesis.
However, these compounds—generated
in the bacterial cytoplasm—have to be
released from the cell through cell disruption processes in order to proceed to the
following separation procedures in the
downstream step. Cell disruption and the
release of all intracellular components are
regularly achieved by different lysis methods, including mechanical (sonication,
French Press, freeze-thaw, homogenization) and non-mechanical (lysozyme and
non-ionic detergents) approaches or different combinations of both.
In the last decades, since the design of
an optimal production process is known
to be a decisive factor regarding the final
yield, quality and application of the
obtained product, an important effort
has been done to develop novel strains
and plasmids, process monitoring, control
strategies and downstream processing, as
well as the application of new strategies of
protein engineering (the use of solubility
Volume 2 Issue 4
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Figure 1. Conventional and optimized cell disruption methods
used
for
and
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and purification tags), and emerging biological principles such as systems biology
and metabolic pathways and key biosynthesis enzymes engineering.12,13
However, in contrast, cell disruption
processes have not been standardized,
being the release of the products still a
delicate step of the whole production process.14 In this context, nowadays there is
still no bacterial cell disruption method
that satisfies the needs of all potential
applications, especially those in which
bacterial materials are used in clinical
applications and need to be free from
potentially toxic bacterial contaminants.
These include conventional protein drugs
but also new particulate materials such as
recombinant engineered PHA granules as
nano-/micro-beads for use in biomedical
applications15 and magnetosomes and IBs,
with application in cell culture and regenerative medicine.9,16-18
Although a diversity of disruption
methods are available, sonication19-21 and
French Press14 are the most widely used
and the most efficient regarding release
of recombinant proteins (Fig. 1). They
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have been widely accepted as appropriate
protocols for the release of noteworthy
yields of soluble proteins without altering
their physicochemical features. Enzymatic
lysis with lysozyme might represent, a
good alternative to mechanical disruption (Fig. 1), although in some cases it has
been described that the use of this enzyme
might perturb the quality of the released
protein.14
On the other hand, since a wide number of proteins of industrial and pharmaceutical interest cannot be produced
in their soluble form, they are recovered
from IBs. Refolding procedures for a more
efficient recovery of correctly folded proteins have significantly improved in the
last decade.22,23 However, the efficiency of
the whole process can be determined not
only by the employed refolding strategy,
but also by the cell lysis method employed
for IB isolation,22,24,25 as it occurs with
soluble proteins. Again, even though most
of the reports describing IB isolation for
further protein refolding use lysozyme
or mechanical methods like sonication
or French press (Fig. 1),26,27 a detailed
Bioengineered Bugs
analysis of such protocols reveals the lack
of a standardized method.27
Furthermore, it is important to point
out that the scene significantly changes
when these IBs are purified to be directly
used as nanoparticles for either catalysis
process as natural biocatalysts28 or as biomaterials able to enhance mammalian cell
proliferation.16-18 Thus, in this situation, it
is necessary to redesign the downstream
processing including cell disruption and
IB release and isolation from cell debris
to preserve not only IB mechanical and
biological stability but also, and not less
important, to obtain fully bacterial free
IBs.10 Consequently, the absence of viable
cell contamination in the final product
becomes a critical parameter to be considered in the development of a cell lysis protocol.10 Since many of the current protocols
for IB isolation usually offer between 95
to 99% of protein recovery, what would
be perfectly fine regarding recovery efficiency, the number of viable bacteria in
the final sample might still be too high,
and incompatible with the above mentioned applications.10 In this context, we
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have recently developed an adapted procedure,10 which combines both chemical and
mechanical disruption methods (Fig. 1).
Specifically, this new protocol includes
the following steps: (1) froze/thaw cycle,
(2) lysozyme treatment (chemical lysis
method), (3) detergent washing, (4) sonication step (mechanical lysis method), (5)
detergent washing, (6) DNase treatment
and (7) final washing step.10 Moreover,
this procedure also includes the determination of viable bacteria in order to ensure
the isolation of bacterial-free protein particles, which could not compromise the
applicability of IBs as biomaterials for
biomedical and industrial applications.10
Besides, Novak and collaborators has also
developed a new technique-based on electrophoretic deposition—for the separation
of IBs from E. coli cells (Fig. 1).29
Regarding the properties of the isolated particles, Peternel and Komel have
recently published a study in which they
describe how the disruption method chosen determines the quality of the isolated
IBs.30 The authors point out that, even
though enzymatic lysis is the gentlest
method towards the obtained product,
high amounts of impurities are found
in the final sample. On the other hand,
although it is observed that most of the
methods based on mechanical disruption allow the isolation of highly pure IB,
these methods can damage the IB structure. However, interestingly, Peternel and
Komel describe high-pressure homogenization as a promising alternative (Fig. 1).30
Therefore, the recently published
results show the importance of using an
appropriate disruption protocol to isolate
active and cell-free bacterial IBs.10,30 This
principle could be exploited for the recovery of other nanoparticulate entities that
might sediment together with remaining
whole cells during conventional downstream protocols, such as intracellular
inclusions of polyhydroxyalkanoates,
metal particles or magnetosomes, and that
are to be used in close intimacy with mammalian cells or other biological interfaces
(Fig. 1).9 For instance PHA, commercially
manufactured through fermentation of
recombinant E. coli,13 has been explored
as a promising, well tolerated by mammalian systems biomaterial for various
tissue-engineering applications including
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cardiovascular
systems
applications
among others.31 Magnetosomes, that are
produced in bacteria by standard culture
procedures,32 can be used in regenerative
medicine, like other magnetic particles,
upon their introduction in mammalian
cells, to create complex tissue structures
by magnetic force engineering.9,33 Again
in this case, fully bacterial-free samples
are required.
In summary, bacterial cell disruption
methodologies needed to recover IBs and
other nanoparticles usable in biomedical
applications would be expected to offer
fully cell-free products, a need that had
not yet been identified in general applications, such as conventional protein production, for which a 95% cell disruption
may be largely acceptable.10
Acknowledgments
The authors appreciate the financial support
to their research through MEC (BFU201017450) and AGAUR (2009SGR-108).
We also appreciate the support from The
Biomedical Research Networking Centre
in Bioengineering,
Biomaterials
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an initiative funded by the VI National
R&D&i Plan 2008–2011, Iniciativa
Ingenio 2010, Consolider Program, CIBER
Actions and financed by the Instituto de
Salud Carlos III with assistance from the
European Regional Development Fund.
Antonio Villaverde has been granted with
an ICREA ACADEMIA award (from
ICREA, Catalonia, Spain).
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