Molecular medicine-Characteristics of Inclusion Bodies

Inclusion bodies can form in the cytoplasm
and in the periplasmic space of E. coli.
Wild-type β-lactamase expressed in E. coli
results in the formation of inclusion bodies
in the periplasm, whereas the protein
expressed without its signal sequence
aggregates in the cytoplasm.
The characteristics of the aggregates depend
on how the protein is expressed.
Different sizes and morphologies have
been observed.Generally, inclusion bodies
appear as dense isomorphous aggregates
of nonnative proteins separated from the
rest of the cytoplasm, but not surrounded
42 Aggregation, Protein
(a)
(b)
Fig. 9 Electron micrographs of (a) cytoplasmic β-lactamase inclusion
bodies in E. coli RB791(pGB1) and (b) purified inclusion bodies from the
same origin (courtesy of G.A. Bowden, A.M. Paredes & G. Georgiou).
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 expressed
in E. coli, the lack of birefringence
indicates that inclusion bodies are not crystalline.
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 μmrespectively, 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 transformed
infrared spectroscopy) have shown
that the insoluble nature of inclusion bodies
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 incorporate
othermolecules, for example, inclusion
bodies from E. coli cells overexpressing
β-lactamase contain only between 35
and 95% intact β-lactamase. The rest consists
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 generally
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 sufficient yield, certain advantages may
accrue. Indeed, aggregation generally prevents
proteolytic attack, except when the
protein coaggregates with a protease. The
formation of inclusion bodies is also an advantage
for the production of proteins that
are toxic for the host cells. Furthermore,
these aggregates contain a great quantity
of the overexpressed protein.