error-prone PCRwhere deliberate and random mutations are introduced by a low-
fidelity PCR amplification reaction. The resulting amplified gene is then translated
and its activity assayed. This has already provided novel evolved enzymes such as a
p-nitrobenzyl esterase which exhibits an unusual and surprising affinity for organic
solvents. This accelerated evolutionary approach to protein engineering has been
useful in the production of novel phage displayed antibodies and in the development
of antibodies with enzymic activities (catalytic antibodies).
6.7 Expression of foreign genes
One of the most useful applications of recombinant DNA technology is the ability to
artificially synthesise large quantities of natural or modified proteins in a host cell
such as bacteria or yeast. The benefits of these techniques have been enjoyed for many
years since the first insulin molecules were cloned and expressed in 1982 (Table 6.3).
Contamination of other proteins such as the blood product factor VIII with infectious
agents has also increased the need to develop effective vectors forin vitroexpression
A A
B C
PCR PCR
5
3
5
5
5
3
3
3
3
3
Denature and anneal PCR products
PCR overlap fragments with 3 ends with primers B and C
Fig. 6.33Construction of a synthetic DNA fragment with a predefined mutation using overlap PCR mutagenesis.
234 Recombinant DNA and genetic analysis
