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الكلية كلية العلوم للبنات     القسم قسم علوم الحياة     المرحلة 7
أستاذ المادة اسراء عدنان ابراهيم البغدادي       20/06/2017 08:26:26
Examples of biotechnological applications
of rDNA technology
In the ?nal part of this chapter we will consider some examples of
the types of products that can be produced using rDNA technology in
biotechnological processes. This is a rapidly developing area for many
biotechnology companies, with large-scale investment in both basic
research and in development to production status. This aspect of gene
manipulation technology is likely to become increasingly important
in the future, particularly in medicine and general healthcare, with
many diverse products being brought to market.
11.4.1 Production of enzymes
The commercial production and use of enzymes is already a well-
established part of the biotechnology industry. Enzymes are used in
brewing, food processing, textile manufacture, the leather industry,
washing powders, medical applications, and basic scienti?c research,
to name just a few examples. In many cases the enzymes are pre-
pared from natural sources, but in recent years there has been
a move towards the use of enzymes produced by rDNA methods,
where this is possible. In addition to the scienti?c problems ofGENETIC ENGINEERING AND BIOTECHNOLOGY 217
producing a recombinant-derived enzyme, there are economic fac-
tors to take into account, and in many cases the cost-bene?t analysis
The preparation of enzymes is a
central part of biotechnology
and ranges from the production
of large amounts of low-cost
preparations for bulk applications
to highly specialised enzymes for
use in diagnostics or other
molecular biology techniques.
makes the use of a recombinant enzyme unattractive. Broadly speak-
ing, enzymes are either high-volume/low-cost preparations for use in
industrial-scale operations or are low-volume/high-value products that
may have a very speci?c and relatively limited market.
There is a nice twist to the gene manipulation story in that some
of the enzymes used in the procedures are now themselves produced
using rDNA methods. Many of the commercial suppliers list recombi-
nant variants of the common enzymes, such as polymerases (partic-
ularly for PCR) and others. Recombinant enzymes can sometimes be
engineered so that their characteristics ?t the criteria for a particular
process better than the natural enzyme, which increases the ?delity
and ef?ciency of the process.
In the food industry, one area that has involved the use of recom-
binant enzyme is the production of cheese. In cheese manufacture,
rennet (also known as rennin, chymase, or chymosin) has been used
as part of the process. Chymosin is a protease that is involved in
the coagulation of milk casein following fermentation by lactic acid
bacteria. It was traditionally prepared from animal (bovine or pig) or
fungal sources. In the 1960s the Food and Agriculture Organisation
of the United Nations predicted that a shortage of calf rennet would
develop as more calves were reared to maturity to satisfy increasing
demands for meat and meat products. Today there are six sources for
natural chymosin -- veal calves, adult cows and pigs, and the fungi
Rhizomucor miehei
,Endothiaparasitica,andRhizomucorpusillus.
Chymosin is now also available as a recombinant-derived preparation
from E. coli, Kluyveromyces lactis, and
Aspergillus niger
. Recombinant
chymosin was ?rst developed in 1981, approved in 1988, and is now
used to prepare around 90% of hard cheeses in the UK.
Although the public acceptance of what is loosely called ‘GM
cheese’ has not presented as many problems as has been the case
with other areas of gene manipulation of foodstuffs, there are still
concerns that need to be addressed. In cheese manufacture, there
are three possible objections that can be raised by those who are
Some aspects of biotechnology,
such as the use of GMOs in food
preparation or modi?cation, may
lead to public concern about the
potential impact on health. This
is an important aspect that we all
have a role in debating.
concerned about GM foods. First, milk could have been produced from
cows treated with recombinant growth hormone (see Section 11.4.2).
Second, the cows could have been fed with animal feeds contain-
ing GM soya or maize. The third concern is the use of recombinant-
derived chymosin. Despite these fears, many consumers are content
that cheese is not itself a genetically modified organism (GMO), but
is the product of a product of a GMO.
A ?nal example of recombinant-derived proteins in consumer
products is the use of enzymes in washing powder. Proteases and
lipases are commonly used to assist cleaning by degradation of pro-
tein and lipid-based staining. A recombinant lipase was developed in
1988 by Novo Nordisk A/V (now known as Novozymes). The company is
the largest supplier of enzymes for commercial use in cleaning appli-
cations. Their recombinant lipase was known as Lipolase TM
, which
was the ?rst commercial enzyme developed using rDNA technology218 GENETIC ENGINEERING IN ACTION
and the ?rst lipase used in detergents. A further development involved
an engineered variant of Lipolase called Lipolase Ultra, which gives
enhanced fat removal at low wash temperatures.
11.4.2 The BST story
Not all rDNA biotechnology projects have a smooth passage from
inception to commercial success. In Section 11.3 we considered some
of the requirements for a biotechnology-based process to be devel-
oped. The story of recombinant bovine somatotropin (rBST) illus-
trates some of the problems that may be encountered once the scien-
ti?c part of the process has been achieved. In bringing a recombinant
product such as rBST (and the examples outlined earlier) to market,
many aspects have to be considered. The basic science has to be carried
out, followed by technology transfer to get the process to a commer-
cially viable stage. Approval by regulatory bodies may be required, and
?nally (and most critical from a commercial standpoint) the product
has to gain market acceptance and establish a consumer base. We can
?nd all of these aspects in the BST story.
BST is also known as bovine growth hormone and is a natu-
rally occurring protein that acts as a growth promoter in cattle. Milk
production can be increased substantially by administering BST and,
thus, it was an attractive target for cloning and production for use in
the dairy industry. The basic science of rBST was relatively straight-
forward, and scientists were already working on this in the early
BST was one of the early
successes of biotechnology, in
that recombinant BST was the
result of achieving the aim of
producing a useful protein by
expressing cloned DNA in a
bacterial host.
1980s. The BST gene was in fact one of the ?rst mammalian genes to
be cloned and expressed, using bacterial cells for production of the
protein. Thus, the production of rBST at a commercial level, involv-
ing the basic science and technology transfer stages, was achieved
without too much dif?culty. A summary of the process is shown in
Fig. 11.7.
With respect to approval of new rDNA products, each country
has its own system. In the USA, the Food and Drug Administra-
tion (FDA) is the central regulatory body, and in 1994 approval was
given for the commercial distribution of rBST, marketed by Monsanto
under the trade name Posilac TM
. At that time the European Union did
not approve the product, but this was partly for socioeconomic rea-
sons (increasing milk production was not necessary) rather than for
any concerns about the science. Evaluation of evidence at that time
suggested that milk from rBST-treated cows was identical to normal
untreated milk, and it was therefore unlikely that any negative effects
would be seen in consumers.
The effects of rBST must be considered in three different contexts --
the effect on milk production, the effects on the animals themselves,
and the possible effects on the consumer. Milk production is usually
increased by around 10--15% in treated cows, although yield increases
of much more than this have been reported. Thus, from a dairy herd
management viewpoint, use of rBST would seem to be bene?cial. How-
ever, as is usually the case with any new development that is aimed
at ‘improving’ what we eat or drink, public concern grew along with
the technology. The concerns fuel a debate that is still ongoing and isGENETIC ENGINEERING AND BIOTECHNOLOGY 219
BST gene
(a)
(b)
(c)
plasmid vector
E. coli host cell
vector
RE
recombinant
rBST
commercial production administration
Fig. 11.7 Production of recombinant bovine growth hormone (rBST). (a) A plasmid
vector is prepared from E. coli and cut with a restriction enzyme. (b) The BST gene
coding sequence is ligated into the plasmid to generate the recombinant, which produces
rBST protein in the cell following transformation. Scale-up to commercial production is
shown in (c) and, with product approval granted, administration can begin. The whole
process from basic science to market usually takes several/many years from start to
?nish, with a large amount of investment capital required. From Nicholl (2000), Cell &
Molecular Biology, Advanced Higher Monograph Series, Learning and Teaching Scotland.
Reproduced with permission.
at times emotive. One area that is hotly debated is the effect of rBST
on the cows themselves. Administering rBST can produce localised
swelling at the site of injection and can exacerbate problems with
foot infections, mastitis, and reproduction. The counter-argument is
that many of these problems occur anyway, even in herds that are
rBST-free. On balance, however, the evidence does suggest that ani-
mal welfare is compromised to some extent when rBST is used.
The possible effects of rBST use on human health is another area
of great concern and debate. The natural hormone (and therefore the
recombinant version also) affects milk production by increasing the220 GENETIC ENGINEERING IN ACTION
levels of insulin-like growth factor (IGF-1), which causes increased
milk production. Administration of rBST generates elevated levels of
IGF-1, and there is evidence that IGF-1 can stimulate the growth of
cancer cells. Thus, the concern is that using rBST could pose a risk
to health. The counter-argument in this case is that the levels of IGF-
1 in the early stages of lactation are higher than those generated
by the use of rBST in cows 100 days after lactation begins, which is
often when it is administered. This arguably means that milk from
early lactating cows should not be drunk at all if there are any con-
cerns about IGF-1. Those who oppose the use of rBST point out that,
unlike a therapeutic protein that would be used for a limited num-
ber of patients, milk is consumed by most people, and any inherent
risk, no matter how small, is therefore unacceptable. On the basis of
Concerns about a biotechnology
product or process are often
multifaceted and can generate
emotive debate; it is sometimes
dif?cult to separate evidence
from speculation.
this uncertainty, many countries have banned the use of rBST, cit-
ing both the animal welfare issue and the potential risk to health as
reasons. The arguments look set to continue into the future as com-
mercial, animal welfare, and human health interests clash. Following
the debate provides an interesting illustration of the problems sur-
rounding the use of gene technology and of the need for objective
assessment of risks and the avoidance of emotive judgements.
11.4.3 Therapeutic products for use in human healthcare
Although the production of recombinant-derived proteins for use in
medical applications does raise some ethical concerns, there is little
serious criticism aimed at this area of biotechnology. The reason is
largely that therapeutic products and strategies are designed to alle-
viate suffering or to improve the quality of life for those who have a
treatable medical condition. In addition, the products are used under
medical supervision, and there is a perception that the corporate
interests that tend to be highlighted in the food debate have less of an
impact in the diagnosis and treatment of disease. In fact, there is just
as much competition and investment risk associated with the med-
ical products ?eld as is the case in agricultural applications; there
does, however, seem to be less emotive debate in this area, so use
in medical applications is therefore apparently much more accept-
able to the public. In addition to the actual treatment of conditions,
the area of medical diagnostics is a large and fast-growing sector of
the biotechnology market, with rDNA technology involved in many
aspects.
Recombinant DNA products for use in medical therapy can be
divided into three main categories. First, protein products may be
Recombinant DNA products for
medical applications are often
more easily accepted by the
public than is the case for GMOs
used in food production.
used for replacement or supplementation of human proteins that
may be absent or ineffective in patients with a particular illness.
Second, proteins can be used in specific disease therapy, to allevi
ate a disease state by intervention. Third, the production of recom-
binant vaccines is an area that is developing rapidly and that offers
great promise. Some examples of therapeutic proteins produced using
rDNA technology are listed in Table 11.1. We will consider examples
from each of these three areas to illustrate the type of approach taken
in developing a therapeutic protein.
The widespread condition diabetes mellitus (DM) is usually caused
either by -cells in the islets of Langerhans in the pancreas failing
to produce adequate amounts of the hormone insulin, or by target
cells not being able to respond to the hormone. Many millions of
people worldwide are affected by DM, and the World Health Organi-
sation estimates that the global incidence will double by 2025. Some
3--6% of people in the UK and the USA have diabetes, although there
is thought to be signi?cant underdiagnosis. Sufferers are classed as
having either type I DM (formerly known as insulin-dependent DM
or IDDM) or type II DM (formerly non-insulin-dependent DM or
NIDDM). Some 10% of patients have type I DM, with around 90% hav-
ing type II. There are also some other variants of the disease that are
much less common. Type I patients obviously require the hormone,
but many type II patients also use insulin for satisfactory control
of their condition. Delivery of insulin is achieved by injection (tra-
ditional syringe or ‘pen’-type devices), infusion using a small pump
and catheter, or inhalation of powdered insulin.
Insulin is composed of two amino acid chains, the A-chain (acidic,
21 amino acids) and B-chain (basic, 30 amino acids). When synthesised
naturally, these chains are linked by a further 30--amino acid peptide
called the C-chain. This 81--amino acid precursor molecule is known
as proinsulin. The A- and B-chains are linked together by disulphide
bonds between cysteine residues, and the proinsulin is cleaved by a
protease to produce the active hormone shown in Fig. 11.8. Insulin
was the ?rst protein to be sequenced -- by Frederick Sanger in the mid
1950s.
As DM is caused by a problem with a normal body constituent
(insulin), therapy falls into the category of replacement or supple-
mentation. Banting and Best developed the use of insulin therapy
in 1921, and for the next 60 or so years diabetics were dependent
Recombinant insulin for use in
the treatment of diabetes is one
of the major success stories of
rDNA-based biotechnology in
that its availability has had a
major impact on the lives of
millions of people.
on natural sources of insulin, with the attendant problems of sup-
ply and quality. In the late 1970s and early 1980s rDNA technology
enabled scientists to synthesise insulin in bacteria, with the ?rst
approvals granted by 1982. Recombinant-derived insulin is now avail-
able in several forms and has a major impact on diabetes therapy.GENETIC ENGINEERING AND BIOTECHNOLOGY 223
A B
A-chain purified from
lactose-induced culture
B-chain purified from
separate culture
A- and B-chains combined
to give functional insulin
Fig. 11.9 Production of
recombinant-derived insulin by
separate fermentations for A- and
B-chains. Lactose is used to induce
transcription of the cloned gene
sequences from the lac promoter.
Following translation, the products
are puri?ed to give A- and B-chains
that are then combined chemically
to give the ?nal product.
One of the most widely used forms is marketed under the name
Humulin TM
by the Eli Lilly Company.
In an early method for the production of recombinant insulin,
the insulin A- and B-chains were synthesised separately in two bacte-
rial strains. The insulin A and B genes were placed under the control
of the lac promoter, so that expression of the cloned genes could be
switched on by using lactose as the inducer. Following puri?cation
of the A- and B-chains, they were linked together by a chemical pro-
cess to produce the ?nal insulin molecule. The process is shown in
Fig. 11.9. A development of this method involves the synthesis of the
entire proinsulin polypeptide (shown in Fig. 11.10) from a single gene
sequence. The product is converted to insulin enzymatically.
There are many recombinant proteins for use in speci?c disease
therapy. One example of this type of protein is tissue plasminogen
activator (TPA). This is a protease that occurs naturally and functions
in breaking down blood clots. TPA acts on an inactive precursor pro-
tease called plasminogen, which is converted to the active form called
S
S
S S
B-chain
S
S
C-chain
A-chain
P
P
Fig. 11.10 Proinsulin. This
molecular precursor of insulin is
synthesised as an 81–amino acid
polypeptide. The C peptide
sequence is then removed by a
protease (P) to leave the A- and
B-chains to form the ?nal insulin
molecule. Proinsulin is now
synthesised intact during
rDNA-based production of insulin.224 GENETIC ENGINEERING IN ACTION
plasmin. This protease attacks the clot by breaking up fibrin, the pro-
tein that is involved in clot formation. TPA is used as a treatment for
Tissue plasminogen activator is
another example of a valuable
therapeutic protein that is
produced by rDNA technology.
heart attack victims. If administered soon after an attack, it can help
reduce the damage caused by coronary thrombosis.
Recombinant TPA was produced in the early 1980s by the com-
pany Genentech using cDNA technology. It was licensed in the USA
in 1987, under the trade name Activase, for use in treatment of acute
myocardial infarction. It was the ?rst recombinant-derived therapeu-
tic protein to be produced from cultured mammalian cells, which
secrete rTPA when grown under appropriate conditions. The amount
of rTPA produced in this way was suf?cient for therapeutic use; thus,
a major advance in coronary care was achieved. Further uses were
approved in 1990 (for acute massive pulmonary embolism) and 1996
(for acute ischaemic stroke).
The ?nal group of recombinant-derived products are vaccines.
There are now many vaccines available for animals, and the devel-
opment of human vaccines is also beginning to have an impact in
healthcare programmes. One vaccine that has been produced by rDNA
methods is the hepatitis B vaccine. The yeast S. cerevisiae is used to
express the surface antigen of the hepatitis B virus (HBsAg), under
the control of the alcohol dehydrogenase promoter. The protein can
then be puri?ed from the fermentation culture and used for inocula-
tion. This removes the possibility of contamination of the vaccine by
blood-borne viruses or toxins, which is a risk if natural sources are
used for vaccine production.
A further development in vaccine technology involves using trans-
genic plants as a delivery mechanism. This area of research and devel-
opment has tremendous potential, particularly for vaccine delivery in
Vaccine delivery by
incorporation into foods is an
elegant concept that has the
potential to bene?t millions of
people in countries where
large-scale vaccination by
traditional methods is dif?cult.
underdeveloped countries where traditional methods of vaccination
may not be fully effective because of cost and distribution problems.
The attraction of having a vaccine-containing banana or tomato is
clear, and development and trials are currently under way for a vari-
ety of plant vaccines.
The use of gene manipulation techniques in the biotechnology
industry is a major developing area of applied science. In addition
to the scienti?c and engineering aspects of the work, the ?nancing
of biotechnology companies is an area that presents its own risks
and potential rewards -- for example, a new drug may take 10--15
years to develop, at a cost of several hundreds of millions of pounds.
The stakes are therefore high, and many ?edgling companies fail
to survive their ?rst few years of operation. Even established and
well-?nanced companies are not immune to the risks associated with
the development of a new and untried product. The next few years
will certainly be interesting for this sector of the applied science
industry.


المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
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