Gene Cloning

260 Gene Cloning
9.4 Some Problems in Obtaining High Level Production of
Proteins in E. coli
Although the basic idea of expressing genes at a high level in E. coli is
straightforward, there are many pitfalls that can occur between obtaining the
cloned gene of interest and producing large amounts of protein from it.
Understanding how to deal with these often requires an understanding of
the underlying biology that causes the problem in the first place. Problems
can arise in essentially three areas: the protein may not be produced at high
levels, or it may be produced at high levels but in an inactive form, or it may
be produced but is toxic to the cells. Toxicity, as discussed in Section 9.3, can
be dealt with by keeping the promoter for the gene which encodes the toxic
protein fully repressed until cells have grown to a high concentration and
then inducing expression for a short period of time to build up a high level of
protein before the cells die or become damaged. The other two problems
have several possible origins, and it is these that we will discuss now.
Low levels of protein
When the gene for a particular protein is highly expressed in E. coli, levels
of the protein can reach astonishingly high levels, to the extent of even
becoming the most abundant protein in the host. However, it is sometimes
the case that even with a protein encoded by a gene which is expressed
from a strong promoter with a good ribosome binding site on a high copy
number plasmid, final levels of the protein are found to be disappointing.
A number of things can cause this, but two in particular are worthy of note
and are easily remedied.
First, the gene may have been cloned from an organism with a different
codon usage to that of E. coli. (For a discussion of codon usage, see Box
8.1). The levels of tRNAs which recognize rare codons are low within the
cell. If a gene from an organism which frequently uses codons which are
rare in E. coli is introduced into E. coli, it may be poorly expressed simply
because the tRNAs required for its efficient translation are present in low
amounts. There are two potential solutions to this problem.
One is to alter the rare codons in the cloned gene to more commonly
used ones, using site-directed mutagenesis (Section 10.5). This can be very
effective, but the drawback with this method is that if a particular gene has
a large number of rare codons within it, it quickly becomes very laborious.
The other solution is to use a strain of E. coli which has been specifically
engineered to express the rare tRNAs at higher levels. Such strains are commercially
available, and can be remarkably successful at increasing the
yields of protein from genes with rare codons.
Q9.4. Would either of these methods alter the amino acid sequence of the
final protein produced?