Aggregation, Protein
43
by a membrane (Fig. 9). They look like
refractile inclusions, which can be easily
recognized by phase contrast microscopy
when large enough. For prochymosin ex-
pressed in
E. coli
,thelackofbirefringence
indicates that inclusion bodies are not crys-
talline. The size distribution of inclusion
bodies has been studied for prochymosin
and interferon-
γ
, and Marston reported
the mean size of particles to be 0.81 and
1
.
28
µ
m respectively, with a relatively high
void fraction. The void volume was about
70% of the total volume for interferon-
γ
and 85% for prochymosin. Structural
characterization studies using ATR-FTIR
(attenuated total reflectance Fourier trans-
formed infrared spectroscopy) have shown
that the insoluble nature of inclusion bod-
ies may be due to their increased levels of
nonnative intramolecular
β
-sheet content.
Inclusion
bodies
consist
mostly
of
the overexpressed recombinant protein,
and
can
contain
little
contaminating
molecules. Thus, they can be used as a
source of relatively pure misfolded protein
when refolding yields the active protein.
However, some amorphous bodies incor-
porate other molecules, for example, inclu-
sion bodies from
E. coli
cells overexpress-
ing
β
-lactamase contain only between 35
and 95% intact
β
-lactamase. The rest con-
sists of a variety of intracellular proteins,
some lipids, and a small amount of nucleic
acids.
Homogeneous
inclusion
bodies
were obtained by expressing
β
-lactamase
without its leader peptide. Under these
conditions, aggregation occurs within the
cytoplasm. The extent of incorporation of
other macromolecules in inclusion bodies
depends upon the overexpressed protein.
The formation of inclusion bodies gen-
erally appears to be a disadvantage, since
it requires the dissolving of the aggregates
in denaturant and subsequent refolding of
the protein. However, when the recovery
of the active product can be obtained with
a suf±cient yield, certain advantages may
accrue. Indeed, aggregation generally pre-
vents proteolytic attack, except when the
protein coaggregates with a protease. The
formation of inclusion bodies is also an ad-
vantage for the production of proteins that
are toxic for the host cells. Furthermore,
these aggregates contain a great quantity
of the overexpressed protein.
4.1.3
Strategies for Refolding Inclusion
Body Proteins
The recovery of the active protein from
inclusion
bodies
is
crucial
for
indus-
trial purposes. In structural proteomics
today, ef±cient production of genetically
engineered proteins is a prerequisite for
exploiting the information contained in
the genome sequences. The strategy to re-
cover active proteins involves several steps
of puri±cation. The ±rst step, the sepa-
ration 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 ±ltration to remove the soluble frac-
tion from the pellet containing inclusion
bodies and cell debris. The most dif±cult
task is to remove the contaminants; this
is achieved by the washing steps, which
commonly utilize EDTA and low concen-
trations of denaturants or detergents such
as Triton X-100, deoxycholate, or octylglu-
coside. 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 con-
centration of denaturant, eliminating the
need for gradient centrifugation. In this
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