Page 259 - Advances in Textile Biotechnology

240 Advances in textile biotechnology

and economics of scale-up (Huang et al., 2007). These factors depend, in
turn, upon the nature of the target protein encoded, their chemical proper-
ties such as size, iso-electric point, stability over various pH and tempera-
ture ranges, and proteolitic resistance as well as the post-translational

modifications required, such as proper-folding, glycosylation, phosphoryla-

tion, formation of disulfide bridges (Rai and Padh, 2001).
Prokaryotic systems are generally easier to handle and are satisfactory
for most purposes. Many microbial species have a strong natural capability
of expressing proteins, which makes them very attractive candidates for the
heterologous production of recombinant proteins. The most successful and
routinely used microbial recombinant protein expression systems mainly
belong to bacterial and fungal species.

10.4.1 Bacteria

Escherichia coli offers a means for the rapid and economical production of
recombinant proteins and is, by far, the most widely employed host for
many reasons: it is well characterized, its genome sequence is known, many
of its biological processes and metabolic pathways are understood and there
are many genetic tools readily available for its manipulation. Indeed, high
growth rates combined with its ability to express high levels of heterologous
proteins (i.e. strains producing up to 30% of their total protein as the
expressed gene product), result in high volumetric productivity. Further-
more, it can grow rapidly to high densities in simple and inexpensive media.
However, there are various limitations for E. coli as an expression system.
Many proteins are complex, containing multiple subunits, cofactors/
prosthetic groups, disulfide bonds, and post-translational modifi cations,

including glycosylation, that are essential for their function. Producing such
complex proteins in E. coli may be quite challenging. In addition, high-level
gene expression levels can make these cells physiologically ill and stress-
sensitive, thus creating a metabolic burden that limits nutrient and oxygen
availability, and the recombinant product can induce various levels of toxic-
ity. For this reason, a compromise in balancing the levels of gene expression
and cell growth needs to be reached to maximize the volumetric recombi-
nant protein productivity. Indeed, bacterial lysis to recover the cytoplasmic
proteins often results in the release of endotoxins, which must be removed
from the final product (Chou, 2007).

Recombinant expressed proteins in E. coli can, in principle, be directed
to three different locations, namely the cytoplasm, the periplasm or the
cultivation medium. Expression in the cytoplasm is normally preferable
because production yields are higher. However, protein degradation is more
likely to occur in the cytoplasm of E. coli than in other compartments
(Makrides, 1996). Proteins expressed in large amounts in the cytoplasm

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