Page 260 - Advances in Textile Biotechnology

Developments in recombinant silk and other elastic protein fi bers 241

often accumulate as soluble aggregates, called inclusion bodies, because of
hydrophobic interactions between exposed patches on unfolded chains or

other unknown specific clustering mechanisms and to inadequate protec-
tion from chaperones. These inclusion bodies are a set of structurally
complex aggregates that can be isolated by differential centrifugation and

provide a useful concentration and purification step in simple plasmid con-
structs and protect target proteins from protease degradation (Swartz,
2001).
Extensive efforts have been made in order to improve the recombinant
expression of heterologous proteins in E. coli and many strategies have
been employed for achieving the high yields demanded:

1 strains defi cient in the most harmful natural proteases belonging to the
genetic bacterial background, such as E. coli BL21 and derivative strains;
2 strains with mutations inhibiting mRNA proteases (such as RNase E,
RNase K and RNase III) that stabilize mRNA and increase the fre-
quency of mRNA translation;
3 strains modified by site-directed mutagenesis or co-expressing rare

tRNAs for avoiding codon bias;
4 the development of protein fusion partners in order to simplify the

purification and expression of recombinant proteins such as His-tag
(polyhistidine tag) or glutathione S-transferase (GST) tag, that can
protect target proteins in vivo from intracellular proteolysis, enhance
solubility, or be used as specifi c expression reporters, as well as increas-
ing expression levels as result of mRNA stabilization (Swartz, 2001,
Sørensen and Mortensen, 2005).

10.4.2 Fungi

Many advantages have made yeast a favored alternative to bacterial systems
for recombinant expression of foreign proteins: these lower eukaryotic
systems include a rich diversity of species and metabolic routes able to
produce high yields in recombinant target proteins secreted to the media,
the availability of the complete genomic sequence and the possibility to
manipulate it, provide a good compromise between bacteria on one side
and mammalian cell lines on the other (Rai and Padh, 2001). However, only
a few, mainly yeast and moulds, have been extensively exploited for their
heterologous protein production skills. Yeast species such as Saccharomy-
ces, Pichia, and Hansenula are well known as efficient gene expression hosts

−1
with good fermentation properties (Pichia pastoris until 65 g L ). Similarly,
mould species such as Trichoderma and the Aspergilli, are distinguished by
−1
their high enzyme secretion levels that can reach 20 g L . Some disadvan-
tages of these expression systems include hypermannosylation, hindering

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