Gene Cloning

Production of Proteins from Cloned Genes 253
Strong promoter
Multiple cloning site
Transcription terminator
Ribosome
binding
site
Origin of
replication
Selectable marker
(e.g. antibiotic resistance)
Figure 9.1 The key features of a typical vector for protein production.
are discussed in detail in the next section. Several other features important
in these plasmids, which are often referred to as expression vectors, are
summarized in Figure 9.1 and discussed in more detail below.
Promoters for expression
Protein production requires the use of a promoter which can be used to
drive expression of the gene to high levels. This promoter will be upstream
of the gene of interest in a suitable plasmid vector, such that it directs transcription
of the gene which encodes the protein of interest. A number of
different promoter systems are widely used in E. coli, with the choice for
any particular experiment depending on a variety of factors. The two most
important features of a promoter are its strength and the degree to which
expression from it can be regulated. The first is important in achieving high
levels of expression: in general, the stronger the promoter, the more mRNA
will be synthesized, and the more protein will result from the translation of
this mRNA. The ability to control expression is also important, however,
particularly as it is often the case that high levels of expression of a heterologous
protein can lead to a decrease in growth of E. coli, or even death.
Thus the ideal promoter for most expression experiments is one which is
strong, but easy to turn down or off.
The simplest examples of promoters for protein expression are those
which are derived from operons of E. coli, with the lac promoter of the lac
operon, and the ara promoter from the arabinose operon (also sometimes
referred to as the pBAD promoter), being widely used (Figure 9.2). You may
already know that expression from the lac promoter is regulated by the lac
repressor protein, LacI (see Chapter 11 for more information on regulation
of gene expression). In the absence of lactose LacI is bound to the DNA,