Sources and upstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 109

Table 5.3 Levels of expression of various biopharmaceuticals produced
in recombinant E. coli cells

Biopharmaceutical Level of expression (% of total cellular protein)

IFN- γ 25

Insulin 20

IFN- β 15

TNF 15

α 1 -Antitrypsin 15

IL-2 10

hGH 5

• heterologous proteins accumulate intracellularly;

• inability to undertake post-translational modifi cations (particularly glycosylation) of proteins;

• the presence of lipopolysaccharide (LPS) on its surface.

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intra-
cellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heter-
ologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular
protein production complicates downstream processing (relative to extracellular production) as:

• additional primary processing steps are required, i.e. cellular homogenization with subsequent

removal of cell debris by centrifugation or fi ltration;

• more extensive chromatographic purifi cation is required in order to separate the protein of inter-

est from the several thousand additional homologous proteins produced by the E. coli cells.

An additional complication of high-level intracellular heterologous protein expression is in-
clusion body formation. Inclusion bodies (refractile bodies) are insoluble aggregates of partially
folded heterologous product. Because of their dense nature, they are easily observed by dark-fi eld
microscopy. Presumably, when expressed at high levels, heterologous proteins overload the normal
cellular protein-folding mechanisms. Under such circumstances, it would be likely that hydropho-
bic patches normally hidden from the surrounding aqueous phase in fully folded proteins would
remain exposed in the partially folded product. This, in turn, would promote aggregate formation
via intermolecular hydrophobic interactions.
However, the formation of inclusion bodies displays one processing advantage: it facilitates
the achievement of a signifi cant degree of subsequent purifi cation by a single centrifugation step.
Because of their high density, inclusion bodies sediment even more rapidly than cell debris. Low-
speed centrifugation thus facilitates the easy and selective collection of inclusion bodies directly
after cellular homogenization. After collection, inclusion bodies are generally incubated with
strong denaturants, such as detergents, solvents or urea. This promotes complete solubilization
of the inclusion body (i.e. complete denaturation of the proteins therein). The denaturant is then
removed by techniques such as dialysis or diafi ltration. This facilitates refolding of the protein, a
high percentage of which will generally fold into its native, biologically active, conformation.