Gene Cloning

Production of Proteins from Cloned Genes 265
first is to direct the protein to a compartment in E. coli where disulfide
bond formation does occur, namely the bacterial periplasm (i.e. the area
between the inner and outer membranes of the bacterial cell). Unlike the
cytoplasm, the periplasm is more or less in equilibrium with the medium
in which the organism is being grown, and this will usually be oxidizing,
thus allowing disulfide bonds to form. Moreover, the bacterial periplasm
(like the eukaryotic endoplasmic reticulum) contains enzymes which help
the formation of disulfide bonds. As discussed in Box 9.2, selected proteins
made in E. coli are transported across the inner membrane to the
periplasm. These proteins are identified as being destined for the
periplasm by the presence of an N terminal “signal sequence”. Fusing the
codons for the signal sequence from a typical bacterial periplasmic protein
to the gene for a heterologous protein will thus cause that protein to be
directed to the periplasm, where disulfide bond formation can occur.
Using precisely this approach, several eukaryotic proteins which contain
large numbers of disulfide bonds have been successfully synthesized in
their active form in E. coli.
The second approach requires an ingenious manipulation of E. coli to
bring about a change in the redox state of its cytoplasm. The details of this
are beyond the scope of this book, but essentially the E. coli cytoplasm is
normally reducing (meaning that the side chains on the cysteines in proteins
will be in the reduced thiol form, -SH) due to the effect of two different
pathways which can be removed by mutating particular genes. When
this is done, the cytoplasm becomes more oxidizing, and so cysteines can
then form disulfide bonds even when the protein is in the cytoplasm.
The points above summarize just some of the issues that can arise when
attempting to maximize protein expression in E. coli, and how to deal with
them. These are also shown in flow diagram format in Figure 9.7.
9.5 Beyond E. coli: Protein Expression in Eukaryotic Systems
Versatile and powerful though E. coli is for expressing proteins, it does have
limitations. Its major drawback arises from the fact that the properties of
proteins often depend not only on the primary amino acid sequence of the
protein but also on modifications to the protein which are made to it after
it has been translated. This is particularly true of eukaryotic proteins,
where the problem is that the modifications made are often ones which
cannot be made in E. coli, simply because it lacks the enzymes to do them.
Probably the most significant post-translational modification is N-
linked glycosylation, which involves the addition of chains of specific sugar
molecules to specific asparagine residues in certain proteins. It takes place
in the endoplasmic reticulum in eukaryotic cells. The chains are further
modified, often very significantly, by the action of different enzymes in the
endoplasmic reticulum and in the Golgi body (Figure 9.8). The molecular
mass of these chains (referred to as glycans) can be quite large, accounting