Biomedical Engineering Reference
In-Depth Information
Box 10.2
continued
position
−
1. The expression of foreign genes in
animals can also be inefficient in some cases due
to suboptimal codon choice, which reflects the fact
that different organisms prefer to use different
codons to specify the same amino acid. If a transgene
contains a codon that is commonly used in the source
organism but rarely used in the host, translation
may pause at that codon due to the scarcity of the
corresponding transfer RNA (tRNA). This will reduce
the rate of protein synthesis and may even lead to
truncation of the protein or frame-shifting. It may
therefore be beneficial to 'codon-optimize'
transgenes for the expression host.
correctly. For example, many proteins intended for
therapeutic use require authentic glycosylation
patterns not only for correct function, but also to
prevent an immune response in the patient. Since
specific types of modification occur in particular cell
compartments, it is necessary to consider strategies
for targeting the recombinant protein to the correct
compartment to ensure that it is appropriately
modified. Proteins that need to be glycosylated, for
example, must be targeted to the secretory pathway
using a
signal peptide
. Many mammalian expression
vectors are available for this purpose, and they
incorporate heterologous signal peptides. The figure
opposite shows the Invitrogen vector pSecTag2,
which incorporates a sequence encoding the murine
immunoglobulin light-chain signal peptide for high-
efficiency targeting to the secretory pathway. Note
also that the C terminus of the recombinant protein is
expressed as a fusion to two different epitope tags to
facilitate protein purification (see p. 76).
The incorporation of a targeting signal
If the goal of an expression study is to recover large
amounts of a functional
eukaryotic
protein, it is
necessary to consider whether that protein needs to
be post-translationally modified in order to function
