Transcript recombinant DNA

10.15. Recombinant DNA
(Genetic Engineering)
prof. aza
10.15. Recombinant DNA (Genetic
Engineering)
• The body requires a constant supply of
certain peptides and proteins if it is to
remain health and function normally.
Many of these peptides and proteins are
only produced in a small quantities.
• They will be produced only if the
correct genes are present in the cell.
prof. aza
• Consequently, if a gene is
missing or defective an
essential protein will not be
produced, which can lead to a
diseased state.
prof. aza
• For example, cystic fibrosis is
caused by a defective gene. This
faulty gene produces a defective
membrane protein, cystic fibrosis
transmembrane regulator (CFTR),
which will not allow the free
passage of chloride ions through
the membrane
prof. aza
• The passage of chloride ions through a
normal membrane into the lungs is
usually accompanied by a flow of water
molecules in the same direction.
• In membranes that contain CFTR the
transport of water through the
membrane into the lungs is reduced.
prof. aza
• This viscous mucus clogs the lungs
and makes breathing difficult, a
classic symptom of cystic fibrosis.
It also provides a breeding ground
for bacteria that cause pneumonia
and other illnesses.
prof. aza
• Several thousand hereditary diseases
found in humans are known to be caused
by faulty genes. Recombinant DNA
(rDNA) technology (genetic engineering)
offers a new way of combating these
hereditary diseases by either replacing
the faulty genes or producing the
missing peptides and proteins so that
they can be given as a medicine (see
section 10.15.2).
prof. aza
• The first step in any use of
recombinant DNA technology is to
isolate or copy the required gene.
There are three sources of the
genes required for cloning.
• The two most important are
genomic and copy or complementary
DNA (cDNA) libraries.
prof. aza
• In the first case the library
consists of DNA fragments
obtained from a cell’s genome,
whilst in the second case the
library consists of DNA fragments
synthesised by using the mRNA for
the protein of interest.
prof. aza
• The third is by the automated
synthesis of DNA, which is only
feasible if the required base
sequence is known.
• This may be deduced from the
amino acid sequence of the required
protein if it is known.
prof. aza
• Once the gene has been obtained it
is inserted into a carrier (vector)
that can enter a host cell and be
replicated, propagated and
transcripted into mRNA by the
cellular biochemistry of that cell.
This process is often referred to
as gene cloning.
prof. aza
• The mRNA produced by the cloned DNA
is used by the cell ribosomes to produce
the protein encoded by the cloned DNA.
In theory, gene cloning makes it possible
to produce any protein provided that it
is possible to obtain a copy of the
corresponding gene. Products produced
using recombinant DNA usually have
recombinant, r or rDNA in their names.
prof. aza
15.1. Gene cloning
• Bacteria are frequently used as
host cells for gene cloning. This is
because they normally use the same
genetic code as humans to make
peptides and proteins.
• However, in bacteria the mechanism
for peptide and protein formation
is somewhat different.
prof. aza
• It is not restricted to the
chromosomes but can also occur in
extranuclear particles called
plasmids.
• Plasmids are large circular
supercoiled DNA molecules whose
structure contains at least one gene
and a start site for replication.
prof. aza
• However, the number of genes
found in a plasmid is fairly limited,
although bacteria will contain a
number of identical copies of the
same plasmid.
• It is possible to isolate the
plasmids of bacterial cells.
prof. aza
• The isolated DNA molecules can be
broken open by cleaving the
phosphate bonds between specific
pairs of bases by the action
enzymes known as restriction
enzymes or endonucleases
prof. aza
• Each of these enzymes, of which
over 500 are known, will only cleave
the bonds between specific
nucleosides.
• For example, EcoRI cleaves the
phosphate link between guanosine
and adenosine whilst Pvu II cuts
the chain between cytidine and
thymine nucleosides.
prof. aza
• Cutting the strand can result in
either blunt ends, where the
endonuclease cuts across both
chains of the DNA at the same
points, or cohesive ends (sticky
ends), where the cut is staggered
from one chain to the other (Fig.
10.47).
prof. aza
• The new non-cyclic structure of the
plasmid is known as linearised DNA
in order to distinguish it from the
new insert or foreign DNA.
prof. aza
• This foreign DNA must contain the
required gene, a second gene system
that confers resistance to a specific
antibiotic and any other necessary
information. It should be remembered
that a eukaryotic gene is made up of
exons separated by introns, which are
sequences that have no apparent use.
prof. aza
• Figure 10.47 (a) Blunt and (b) cohesive
cuts with compatible adhesive cuts
prof. aza
• Mixing the foreign DNA and the
linearised DNA in a suitable medium
results in the formation of extended
plasmid loops when their ends come into
contact (Fig. 10.48).
• This contact is converted into a
permanent bond by the catalytic action
of an enzyme called DNA ligase.
prof. aza
• Figure
10.48. A
representa
tion of the
main steps
in the
insertion
of a gene
into a
plasmid
prof. aza
• When the chains are cohesive the
exposed single chains of new DNA
must contain a complementary base
sequence to the exposed ends of
the linearised DNA.
prof. aza
• The hydrogen bonding between these
complementary base pairs tends to bind
the chains together prior to the action
of the DNA ligase, hence the name
‘‘sticky ends’’.
• The new DNA of the modified plasmid is
known as recombinant DNA (rDNA).
prof. aza
• However, the random nature of the
techniques used to form the modified
plasmids means that some of the
linearised DNA reforms the plasmid
without incorporating the foreign DNA,
that is, a mixture of both types of
plasmid is formed.
prof. aza
• The modified plasmids are
separated from the unmodified
plasmids when they are reinserted
into a bacterial cell.
prof. aza
• The new plasmids are reinserted into
the bacteria by a process known as
transformation.
• Bacteria are mixed with the new
plasmids in a medium containing calcium
chloride. This medium makes the
bacterial membrane permeable to the
plasmid.
prof. aza
• However, not all bacteria will take
up the modified plasmids.
• Such bacteria can easily be
destroyed by specific antibiotic
action since they do not contain
plasmids with the appropriate
protecting gene.
prof. aza
• This makes isolation of the bacteria
with the modified plasmids
relatively simple.
• These modified bacteria are
allowed to replicate and, in doing so,
produce many copies of the
modified plasmid.
prof. aza
• Under favourable conditions one
modified bacterial cell can produce
over 200 copies of the new plasmid.
The gene in these modified
plasmids will use the bacteria’s
internal machinery to automatically
produce the appropriate peptide or
protein.
prof. aza
• Since many bacteria replicate at a
very rapid rate this technique
offers a relatively quick way of
producing large quantities of
essential naturally occurring
compounds that cannot be produced
by other means.
prof. aza
• Plasmids are not the only vectors
that can be used to transport DNA
into a bacterial host cell.
• Foreign DNA can also be inserted
into bacteriophages and cosmids by
similar techniques.
prof. aza
• Bacteriophages (phage) are viruses that
specifically infect bacteria whilst a
cosmid is a hybrid between a phage and
a plasmid that has been especially
synthesised for use in gene cloning.
Plasmids can be used to insert
fragments containing up to 10
kilobasepairs (kbp), phages up to 20 kbp
and cosmids 50 or more kbp.
prof. aza
• It is not always necessary to use a
vector to place the recombinant
DNA in a cell. If the cell is large
enough, the recombinant DNA may
be placed in the cell by using a
micropipette whose overall tip
diameter is less than 1 mm.
prof. aza
• Only a small amount of the
recombinant DNA inserted in this
fashion is taken up by the cell’s
chromosomes.
• However, this small fraction will
increase to a significant level as the
cell replicates (Fig. 10.48).
prof. aza
• Host cells for all methods of cloning are
usually either bacterial or mammalian in
origin. For example, bacterial cells often
used are E. coli and eukaryotic yeast
while mammalian cell lines include
Chinese hamster ovary (CHO), baby
hamster kidney (BHK) and African green
monkey kidney (VERO).
prof. aza
• In all cases small-scale cultures of
the host cell plus vector are grown
to find the culture containing the
host with the required gene that
gives the best yield of the desired
protein.
prof. aza
• Once this culture has been
determined the process is scaled up
via a suitable pilot plant to
production level (see section 16.6).
The mammalian cell line cultures
normally give poorer yields of the
desired protein.
prof. aza
15.2.2 Manufacture of Pharmaceuticals
• The body produces peptides and
proteins, often in extremely small
quantities, which are essential for its
well being.
• The absence of the necessary’ genes
means that the body does not produce
these essential compounds, resulting in
a deficiency disease that is usually’
fatal.
prof. aza
• Treatment by supplying the patient
with sufficient amounts of the
missing compounds is normally
successful.
• However, extraction from other
natural sources is usually’ difficult
and yields are often low.
prof. aza
• For example, it takes half a million
sheep brains to produce 5mg of
somatostatin a growth hormone
that inhibits secretion of the
pituitary growth hormone.
prof. aza
• Furthermore, unless the source of
the required product is donated
blood there is a limit to the number
of cadavers available for the
extraction of compounds suitable
for use in humans.
prof. aza
• Moreover, there is also the danger that
compounds obtained from human
sources may be contaminated by’ viruses
such as HIV, hepatitis, Creutzfeld–
Jakob disease (mad cow disease) and
others that are difficult to detect.
Animal sources have been used but only
a few human protein deficiency
disorders can be treated with animal
proteins.
prof. aza
• Gene cloning is used to obtain human
recombinant proteins. However, some
proteins will also need post—
translational modification such as
glycosylation and/or the modification of
amino acid sequences.
• These modifications may require
forming different section, of the
peptide chain in the culture medium and
chemically’ combining these sections in
vitro.
prof. aza
• The genes required for these processes
are synthesised using the required peptide
as a blueprint. For example, human
recombinant insuline may he produced in
this manner (Figure 10.12). The genes for
the A and B chains of insulin were
synthesised separately.
prof. aza
• They were cloned separately, using
suitable plasmids. into two different
bacterial strains. One of these strains
is used to produce the A chain whilst
the others is used to produce the B
strain. The chains are isolated and
attached to each other by in vitro
disulphide bond formation.
prof. aza
• This last step is inefficient and
human recombinant insulin is now
made by forming recombinant
proinsulin by gene cloning.
• The proinsulin is converted to
recombinant insulin by proteolytic
cleavage
prof. aza
Figure 10.42. An outline of the synthesis of
recombinant human insulin.
prof. aza