44
Aggregation, Protein
case, the costs of production are consider-
ably reduced.
A variety of techniques are available
to solubilize puriFed inclusion bodies.
The most commonly used solubilizing
reagents are strong denaturants such as
guanidine hydrochloride and urea. Gener-
ally, high denaturant concentrations are
employed, 4 to 6 M for guanidine hy-
drochloride, and 5 to 10 M for urea to
allow the disruption of noncovalent in-
termolecular interactions. Conditions may
differ somewhat according to the denat-
urant and the protein. Lower denaturant
concentrations have been used to solubi-
lize 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 concentra-
tions, 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 and may cause
irreversible chemical modiFcations.
Detergents such as sodium dodecylsul-
fate (SDS) and n-cetyl trimethylammo-
nium bromide (CTAB), have also been
used to solubilize inclusion bodies. Ex-
tensive washing may then be
needed
to
remove the
solubilizing detergents.
They also may be extracted from the re-
folding mixture by using cyclodextrins,
linear dextrins, or cycloamylose. Recent
developments include the use of high
hydrostatic pressure (1–2 kbar) for solu-
bilization and renaturation. ±or proteins
with disulFde bonds, the addition of a re-
ducing reagent such as dithiothreitol or
β
-mercaptoethanol is necessary to disrupt
the incorrectly paired disulFde 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 ac-
tive 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, diaFltration, gel Fltra-
tion, 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 re-
folding of recombinant proteins, because
large downstream processing volumes in-
crease the cost of products. Although
dialysis
through
semipermeable
mem-
branes
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 dial-
ysis, the protein will remain too long
at a critical concentration of denaturant
and aggregate. The removal of the de-
naturant may be accomplished through
gel
Fltration.
However,
here
again,
a
possible aggregation could lead to flow
restriction within the column. DialFltra-
tion through a semipermeable membrane
allows the removal of denaturant and
other small molecules and retains the pro-
tein. This procedure has been used for
large-scale processing and was particularly
efFcient in the refolding of prorennin and
interferon-
β
.
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