Biomedical Engineering Reference
In-Depth Information
approach to cloning has now come to the fore, as it is faster and more convenient than the more
classical methods described above. The process begins by extraction of total genomic DNA from
the source of interest (e.g. human cells if you wish to clone a specifi c human gene). Oligonucleotide
primers whose sequences fl ank the target gene/DNA segment are synthesized and used to amplify
that portion of DNA selectively. Recognition sites for REs can be incorporated into the oligonucle-
otides to allow cloning of the amplifi ed gene, as outlined earlier. Because the target gene sequence
is the only segment of the extracted DNA to be amplifi ed by the prior PCR step, the vast majority of
clones in the library now generated should contain the desired gene. This can be confi rmed by direct
sequencing of the inserted DNA fragment from several of the colonies. Sequencing is important not
only to prove defi nitively that the cloned DNA is the target gene, but also that its sequence perfectly
matches the published sequence. The PCR process is prone to the introduction of sequence errors.
3.4.3 Expressionvectors
The vectors described thus far have been designed to facilitate the cloning of genomic DNA/
cDNA sequences, ultimately in order to identify and isolate a gene/cDNA coding for a particular
polypeptide. The genetic construction of these vectors normally does not support the actual ex-
pression (i.e. transcription and translation) of the gene. Once the gene/cDNA coding for a potential
target protein has been isolated, the goal usually becomes one of achieving high levels of expres-
sion of this target gene. This process entails ligation of the gene into a vector that will support
high-level transcription and translation. In addition to the basic vector elements (e.g. an origin of
replication and a selectable marker, such as an antibiotic resistance gene), expression vectors also
contain all the genetic elements required to support transcription and translation, as described
earlier in this chapter (e.g. promoters, translational start and stop signals, etc.; see Figures 3.7 and
3.8). A wide range of such expression vectors is now commercially available and, obviously, each
is tailored to work best in a specifi c host cell type (e.g. bacterial, yeast, mammalian, etc.). The
choice of exact host cell type in which to express a recombinant therapeutic protein will depend
upon a number of factors, as described in Chapter 5.
3.4.4 Proteinengineering
The advent of rDNA technology renders straightforward the manipulation of a protein's amino
acid sequence. This process, termed site-directed mutagenesis or protein engineering, entails the
controlled alteration of the nucleotide sequence coding for the polypeptide of interest such that
specifi c, predetermined changes in amino acid sequence are introduced. Such changes can include
insertions, deletions or substitutions. Site-directed mutagenesis is now most often undertaken by
using a variant of the basic PCR method already described (Figure 3.15), known as 'overlap PCR',
in which primers of altered nucleotide sequences are used for the PCR reactions.
Protein engineering facilitates a greater understanding of the link between a polypeptide's
amino acid sequence and its structure. It also provides a powerful method of studying the relation-
ship between structure and its function. As such, this technique will help greatly in achieving the
much pursued, but still distant, objective of
de novo
protein design. Protein engineering is also
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