Aggregation, Protein
During the refolding process, the for-
mation of incorrectly folded species and
aggregates usually decreases the refolding
yield. For disul±de-bridged proteins, the
renaturation buffer must contain redox-
shuffling mixture to allow the formation
of correctly paired disul±de bridges. Stabi-
lizing reagents may be added to improve
the refolding yield. An ef±cient strategy
is the addition of small molecules to sup-
press intermolecular interactions leading
to aggregation. Sugar, alcohols, polyols
(including sucrose, glycerol, polyethylene
glycol, isopropanol), cyclodextrin, lauryl-
maltoside, sulfobetains,
low concentrations of denaturants and de-
tergents, have been used to increase the
refolding yield. L-arginine at a concentra-
tion ranging from 0.4 M to 0.8 M is the
most widely used additive today.
Another important factor in the refold-
ing process is the rate of removal of the
denaturant. Since there is kinetic compe-
tition between the correct folding and the
formation of aggregates from a folding in-
termediate, conditions that favor folding
over the accumulation of aggregates must
be found. To optimize this selection, Vil-
ick and de Bernadez–Clark developed a
strategy for achieving high protein refold-
ing yields. They start from a model of
refolding, develop the equations of refold-
ing kinetics, characterize the rate-limiting
step of the process, determine the influ-
ence of various environmental parameters,
and ±nally optimize the system of equa-
tions in a scheme involving dia±ltration to
remove the denaturant. The approach was
evaluated in the refolding of carbonic an-
hydrase from 8 M urea. The yield obtained
after three dia±ltration 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 system for refolding chro-
matography that utilizes GroEL, DsbA, and
peptidyl–prolyl isomerase immobilized on
an agarose gel. Kohler and coworkers
have built a chaperone-assisted bioreac-
tor; 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 de-
sired protein; this can greatly increase
the soluble yield of aggregation-prone pro-
teins. Fusion proteins have also been used
to minimize aggregation.
The recovery of active proteins from in-
clusion 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 pro-
cesses for recombinant proteins have been
introduced. They consist of the introduc-
tion of combinatorial protein engineering
to generate molecules highly speci±c to
a particular ligand. Such methods, which
allow ef±cient recovery of a recombinant
protein, will be increasingly used in indus-
trial scale bioprocesses as well.
The Formation of Amyloid Fibrils and its
Pathological Consequences
The formation of amyloid ±brils plays a key
role in the origin of several neurodegen-
erative pathologies, such as spongiform
ease. Historically, the term amyloid was
introduced to describe ±brillar protein
deposits associated with diseases known
that involve the extracel-
lular deposition of amyloid ±brils and
plaques with the aspect of starch. For
many of these diseases, the major ±brillar
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