Gene Cloning

262 Gene Cloning
Figure 9.6 Inclusion bodies in E. coli, viewed by transmission electron
microscopy (TEM). Image courtesy of Professor Jonathan King, MIT.
under carefully controlled conditions that allow it to reach its native and
active state. Many commercially produced proteins are in fact made in this
way. However, the refolding process can be difficult and expensive, and
sometimes it is preferable to find a way of getting the protein soluble and
correctly folded while still inside the cell. Sometimes the solution to this is
straightforward: the problem arises from very high protein expression, so
the answer is to lower protein expression levels. This may be done by using
a weaker promoter, a lower copy number plasmid vector, or simply by
growing the strains at a lower temperature or in a less rich medium. While
all these have the undesirable effect of also decreasing yield, it is better to
have a small amount of active protein than a large amount of inactive protein.
However, other approaches can also be used.
One approach to the inclusion body problem is to manipulate the folding
capacity of the cell. E. coli (and all other organisms) contains a number
of proteins called molecular chaperones whose role it is to assist protein
folding in various ways. By co-expressing these molecular chaperones at
higher levels when the heterologous proteins are being produced, in some
cases the formation of inclusion bodies is lowered or even stops altogether,
without any decrease in the level of expression of the protein concerned.
Another approach which is sometimes successful is to change the gene
for the protein by adding to it some codons which code, in the same reading
frame as the protein of interest, for a part of another protein which is
very soluble. This is a specific example of a more general method, which