Aggregation, Protein
41
allowed their identifcation. Figure 3 illus-
trates the ±olding pathway o± the protein.
The native protein is highly thermostable
with a T
m
o± 88
C; it is also resistant
to detergents and proteases. During the
in vivo
±olding process, the intermediates
are sensitive to these ±actors, allowing
their identifcation. At low temperature,
almost 100% o± the newly synthesized
chains reach the native trimer con±orma-
tion. When the temperature increases in
the cells, the number o± polypeptide chains
achieving the native state decreases. At
39
C, the maturation proceeds with 30%
e±fciency, while the remainder aggregates
into inclusion bodies. It has been shown
that the aggregation does not result ±rom
an intracellular denaturation o± the na-
tive protein, but is generated ±rom an early
thermolabile intermediate. The aggregated
chains cannot recover their proper ±olding
by lowering the temperature. But when
polypeptide chains that have been synthe-
sized at high temperatures are shi±ted to
low temperature early enough, they can
re±old correctly.
A set o± mutations that alter protein
±olding without modi±ying the proper-
ties and stability o± native P22 tailspike
has been identifed; they are re±erred
to as temperature-sensitive ±olding (ts±)
mutants. These mutations have been sup-
posed to destabilize the already thermola-
bile intermediate and are located at more
than 30 sites in the central region o±
the polypeptide chain. Starting ±rom mu-
tants kinetically blocked in their ±olding, a
second set o± mutants capable o± correct-
ing the ±olding de±ects was selected, and
the sequences surrounding the suppressor
mutations were identifed. Only two sub-
stitution positions on the 666 amino acids
o± the polypeptide chain were su±fcient to
prevent inclusion body ±ormation. Thus,
single temperature mutations that a±±ect
the ±olding pathway but not the native
con±ormation o± a protein are e±fcient in
preventing o±±-pathway and subsequent ag-
gregation. A similar result has been ±ound
±or heterodimeric luci±erase. For recom-
binant proteins such as inter±eron-
γ
and
interleukin 1
β
,a
sw
e
l
la
o
rP
2
2t
a
i
l
-
spike, amino acid substitutions that can
decrease or increase the ±ormation o± in-
clusion bodies without alteration o± the
±unctional structure were ±ound by Wetzel
and coworkers.
The ±ormation o± inclusion bodies is
±requently observed in the production o±
recombinant proteins. High levels o± ex-
pression o± these proteins result in the
±ormation o± inactive amorphous aggre-
gates, and has been reported ±or proteins
expressed in
E. coli
and also in several
host cells, gram-negative as well as gram-
positive bacteria, and eukaryotic cells such
as
Saccharomyces cerevisiae
, insect cells,
and even animal cells. The production
o± recombinant proteins, among them
human insulin, inter±eron-
γ
,in
te
r
leuk
in
1
β
,
β
-lactamase, prochymosin, tissue plas-
minogen activator, basic fbroblast growth
hormone, and somatotropin, gives rise to
inclusion bodies.
4.1.2
Characteristics of Inclusion Bodies
Inclusion bodies can ±orm in the cytoplasm
and in the periplasmic space o±
E. coli
.
Wild-type
β
-lactamase expressed in
E. coli
results in the ±ormation o± inclusion bodies
in the periplasm, whereas the protein
expressed
without
its
signal
sequence
aggregates in the cytoplasm.
The characteristics o± the aggregates de-
pend on how the protein is expressed.
Di±±erent sizes and morphologies have
been observed. Generally, inclusion bodies
appear as dense isomorphous aggregates
o± nonnative proteins separated ±rom the
rest o± the cytoplasm, but not surrounded
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