Page 258 - Advances in Textile Biotechnology

Developments in recombinant silk and other elastic protein fi bers 239

10.4 Expression systems
When recombinant DNA techniques are used to express certain genes
outside their natural expression habitat, the process is described as heter-
ologous gene expression. In order to express a target protein heterologously
in a host, the target DNA-coding sequence has to be known and a suitable
transformation and selection strategy has to exist (Mahmoud, 2007). When
a designed gene is introduced into a suitable vehicle (called a vector; usually
on a plasmid), and then into a selected host, the progeny of such a hosting

cell (called a clone), produce the recombinant protein specified by the

codified gene (Jackel et al., 2008).
For DNA delivery and integration non-viral gene transfer remains the
preferred approach to generate stable cell lines for manufacturing purposes.
Calcium phosphate transfection, electroporation, lipofection and biolistic-
and polymer-mediated gene transfer, are routinely used and are all reason-
ably efficient and reliable procedures for transferency (Godbey and Mikos,

2001, Jo and Tabata, 2008).
Expression constructs are chimerical structures in which the transgene is
bracketed by various regulatory elements. To achieve high yields, the design
of expression constructs must optimize all stages of gene expression, from
transcription to protein stability. The central elements essential in the design
of any recombinant expression systems must include:
1 origin of replication (ori), which is a particular sequence at which rep-
lication is initiated and that allows the autonomous replication of the
vector within the cell;
2 selection markers are sequences encoding a selectable marker that
confers particular features to the transformed hosts for subsequent
selection and assures maintenance of the vector into it;
3 a promoter that controls the level of gene expression. Expression vectors
generally use a strong viral or cellular promoter/enhancer to drive the
expression of the recombinant gene;
4 a terminator, which is a sequence for ensuring that the RNA polymerase
disengages and does not continue to transcribe downstream genes. A
strong transcriptional terminator should be used with a strong promoter;
5 polylinkers or cloning sites for cutting and pasting of DNA fragments
to simplify the insertion of the heterologous gene in the correct orienta-
tion within the cell (Rai and Padh, 2001).

A variety of surrogate hosts are available for in vivo heterologous recom-

binant protein production. These actually encompass five groups of organ-
isms: bacteria, fungi, plants, insects and mammals (Makrides, 1996). The

optimal expression system for generating sufficient amounts of one protein
must be selected based on protein yield, purity, structure, fi nal application

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