Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 724

rate if the consumption rate can be estimated on-line or predicted. However, E. coli is not
a perfect host. The major problems result from the fact that E. coli does not normally secrete
proteins. When proteins are retained intracellularly and produced at high levels, the amount
of soluble active protein present is usually limited due to either proteolytic degradation or
insolubilization into inclusion bodies .
The production of large amounts of foreign protein may trigger a heat-shock response.
One response of the heat-shock regulon is increased proteolytic activity. In some cases,
intracellular proteolytic activity results in product degradation at a rate nearly equal to
the rate of production. More often, the target protein forms an inclusion body. Although
the heterologous protein predominates in an inclusion body, other cellular material is
also often included. The protein in the inclusion body is misfolded. The misfolded
protein has no desirable biological activity and is thus of no value. If the inclusion
bodies are recovered from the culture, the inclusion bodies can be resolubilized and
the activity (and value) can be restored. Resolubilization can vary tremendously in diffi-
culty from one protein to another. When resolubilization is straightforward and recov-
eries are high, the formation of inclusion bodies can be advantageous, as it simplifies
the initial steps of recovery and purification. It is important that during resolubilization
the protein be checked by several analytical methods to ensure that no chemical modi-
fications have occurred. Even slight changes in a side group can alter the effectiveness
of the product.
Other consequences of cytoplasmic protein production can be important. The intracellular
environment in E. coli might not allow the formation of disulfide bridges. Also the protein
will usually start with a methionine, whereas that methionine would have been removed
in normal posttranslational processing in the natural host cell. If the product is retained intra-
cellularly, then the cell must be lysed during recovery. Lysis usually results in the release of
endotoxins (or pyrogens) from E. coli. Endotoxins are lipopolysaccharides (found in the outer
membrane) and can result in undesirable side effects (e.g. high fevers) and death. Thus, puri-
fication is an important consideration.
Many of the limitations on E. coli can be circumvented with protein secretion and excre-
tion. Secretion is defined here as the translocation of a protein across the inner membrane
of E . coli. Excretion is defined as release of the protein into the extracellular compartment.
About 20% of all protein in E. coli is translocated across the inner membrane into the periplas-
mic space or incorporated into the outer membrane. As we have learned in Chapter 10,
secreted proteins are made with a signal or leader sequence. The presence of a signal sequence
is a necessary (but not sufficient) condition for secretion. The signal sequence is a sequence of
amino acids attached to the mature protein, and the signal sequence is cleaved during
secretion.
Many benefits are possible if a protein is secreted. Secretion eliminates an undesired
methionine from the beginning of the protein. Secretion also offers some protection from
proteolysis. Periplasmic proteases exist in E. coli but usually at a low level. They are most
active in alkaline environments. With pH control the target protein degradation can be
reduced. The environment in the periplasmic space promotes the correct protein folding in
some cases (including the formation of disulfide bridges). Proteins in the periplasmic space
can be released by gentle osmotic shock, so that fewer contaminating proteins are present
than if the whole cell was lysed.