The recovery of the active Protein from
inclusion bodies is crucial for industrial
purposes. In structural proteomics
today, efficient production of genetically
engineered proteins is a prerequisite for
exploiting the information contained in
the genome sequences. The strategy to recover
active proteins involves several steps
of purification. The first step, the separation
of the inclusion bodies from the
cell, consists of cell lysis monitored either
by high-pressure homogeneization, or by
a combination of mechanical, chemical,
and enzymatic techniques such as the use
of EDTA and lysozyme. The lysates are
then treated by low-speed centrifugation
or filtration to remove the soluble fraction
from the pellet containing inclusion
bodies and cell debris. The most difficult
task is to remove the contaminants; this
is achieved by the washing steps, which
commonly utilize EDTA and low concentrations
of denaturants or detergents such
as Triton X-100, deoxycholate, or octylglucoside.
Using centrifugation in a sucrose
gradient, it is generally possible to remove
cell debris and membrane proteins. When
the accumulation levels of aggregates are
very high, inclusion bodies may be directly
solubilized by treatment in a high concentration
of denaturant, eliminating the
need for gradient centrifugation. In this
44 Aggregation, Protein
case, the costs of production are considerably
reduced.
A variety of techniques are available
to solubilize purified inclusion bodies.
The most commonly used solubilizing
reagents are strong denaturants such as
guanidine hydrochloride and urea. Generally,
high denaturant concentrations are
employed, 4 to 6 M for guanidine hydrochloride,
and 5 to 10 M for urea to
allow the disruption of noncovalent intermolecular
interactions. Conditionsmay
differ somewhat according to the denaturant
and the protein. Lower denaturant
concentrations have been used to solubilize
cytokines from E. coli inclusion bodies.
The purity of the solubilized protein was
much higher at 1.5 to 2 M guanidinium
chloride than at 4 to 6 M guanidinium
chloride. At higher denaturant concentrations,
contaminating proteins were also
released from the particulate fractions.
Extremes of pH have also been used to
solubilize inclusion bodies and for growth
hormone, proinsulin, and some antifungal
recombinant peptides. However, exposure
to very low or very high pH may not be
applicable to many proteins andmay cause
irreversible chemical modifications.
Detergents such as sodium dodecylsulfate
(SDS) and n-cetyl trimethylammonium
bromide (CTAB), have also been
used to solubilize inclusion bodies. Extensive
washing may then be needed
to remove the solubilizing detergents.
They also may be extracted from the refolding
mixture by using cyclodextrins,
linear dextrins, or cycloamylose. Recent
developments include the use of high
hydrostatic pressure (1–2 kbar) for solubilization
and renaturation. For proteins
with disulfide bonds, the addition of a reducing
reagent such as dithiothreitol or
β-mercaptoethanol is necessary to disrupt
the incorrectly paired disulfide bonds. The
concentrations generally used are 0.1 M
for dithiothreitol and 0.1 to 0.3 M for β-
mercaptoethanol.
When expression levels are very high,
an in situ solubilization method can be
used. It consists of adding the solubilizing
reagent directly to the cells at the end
of the fermentation process. The main
disadvantage of this technique concerns
the release of contaminants.
The last step is the recovery of the active
protein. When inclusion bodies have
been solubilized, the refolding is achieved
by removal of the denaturant. This can
be done by different techniques including
dilution, dialysis, diafiltration, gel filtration,
chromatography, or immobilization
on a solid support. Dilution has been
extensively used. It considerably reduces
concentrations of both denaturant and
protein. This procedure, however, cannot
be applied to the commercial scale refolding
of recombinant proteins, because
large downstream processing volumes increase
the cost of products. Although
dialysis through semipermeable membranes
has been used successfully to
refold several proteins, it is not employed
in large-scale processes. This is because
it requires very long processing times,
and there is the risk that during dialysis,
the protein will remain too long
at a critical concentration of denaturant
and aggregate. The removal of the denaturant
may be accomplished through
gel filtration. However, here again, a
possible aggregation could lead to flow
restriction within the column. Dialfiltration
through a semipermeable membrane
allows the removal of denaturant and
other small molecules and retains the protein.
This procedure has been used for
large-scale processing and was particularly
efficient in the refolding of prorennin and
interferon-β.
Aggregation, Protein 45
During the refolding process, the formation
of incorrectly folded species and
aggregates usually decreases the refolding
yield. For disulfide-bridged proteins, the
renaturation buffer must contain redoxshuffling
mixture to allow the formation
of correctly paired disulfide bridges. Stabilizing
reagents may be added to improve
the refolding yield. An efficient strategy
is the addition of small molecules to suppress
intermolecular interactions leading
to aggregation. Sugar, alcohols, polyols
(including sucrose, glycerol, polyethylene
glycol, isopropanol), cyclodextrin, laurylmaltoside,
sulfobetains, L-arginine, and
low concentrations of denaturants and detergents,
have been used to increase the
refolding yield. L-arginine at a concentration
ranging from 0.4 M to 0.8 M is the
most widely used additive today.
Another important factor in the refolding
process is the rate of removal of the
denaturant. Since there is kinetic competition
between the correct folding and the
formation of aggregates from a folding intermediate,
conditions that favor folding
over the accumulation of aggregates must
be found. To optimize this selection, Vilick
and de Bernadez–Clark developed a
strategy for achieving high protein refolding
yields. They start from a model of
refolding, develop the equations of refolding
kinetics, characterize the rate-limiting
step of the process, determine the influence
of various environmental parameters,
and finally optimize the system of equations
in a scheme involving diafiltration to
remove the denaturant. The approach was
evaluated in the refolding of carbonic anhydrase
from 8 Murea. The yield obtained
after three diafiltration experiments was
69% whereas the model predicted a yield
of 73%.
The properties of molecular chaperones
have also been utilized to increase the
refolding yield. Altamiro and coworkers
have developed a systemfor refolding chromatography
that utilizes GroEL, DsbA, and
peptidyl–prolyl isomerase immobilized on
an agarose gel. Kohler and coworkers
have built a chaperone-assisted bioreactor;
however, it could only be used for
three cycles of refolding and needs to be
improved. Another strategy consists of the
co-overproduction of the DnaK–DnaJ or
GroEL–GroES chaperones with the desired
protein; this can greatly increase
the soluble yield of aggregation-prone proteins.
Fusion proteins have also been used
to minimize aggregation.
The recovery of active proteins from inclusion
bodies is a rather complex process.
Although some general strategies have
been developed, optimal conditions have to
be determined for each protein. Recently,
genetic strategies to improve recovery processes
for recombinant proteins have been
introduced. They consist of the introduction
of combinatorial protein engineering
to generate molecules highly specific to
a particular ligand. Such methods, which
allow efficient recovery of a recombinant
protein, will be increasingly used in industrial
scale bioprocesses as well