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Biotechnology Unit
Plant Engineering for the Production of Valuable Proteins
One of the best-known examples of modern plant biotechnology
lies in genetic transformation whereby selected genes are inserted into
the plant. The genes concerned are normally those that confer upon the plant
various desired agronomic characteristic such as disease or pest resistance.
This is not unlike what can be achieved in conventional plant breeding.
However, large numbers of genes are reshuffled in plant breeding. While
a progeny from sexual crossing might gain a desired trait, it might, at
the same time, also inherit unwanted characteristics or lose some of the
good traits displayed by its parents. In genetic transformation, on the
other hand, the gene controlling a specific trait is inserted into the plant,
leaving the plant's other characteristics generally unaltered. Another principal
difference between conventional breeding and genetic transformation is that,
in the former, the introduction of new genes is limited to those from closely
related species. With genetic transformation, the species barrier can be,
and often is, traversed. For this reason, a genetically transformed plant
is also known as a transgenic plant.
Besides improving crop productivity, genetic transformation
technique has another promising application that is less publicised. DNA
building blocks, called nucleotides, that are present in genes are arranged
in sequences that serve as codes for the synthesis of various proteins.
The immediate product of gene function is the protein that the gene encodes.
Hence, if the gene that controls a foreign protein, such as a pharmaceutical
protein, were inserted into the plant, the latter would then possess the
blueprint to synthesise this foreign protein. Along these lines, transgenesis
has the potential to turn crop plants into living factories for the production
of commercially valuable proteins such as peptide-based pharmaceuticals.
Proteins that are produced through the process of transgenesis are known
as recombinant proteins.
Harvesting foreign proteins from transgenic microorganisms,
animals and plants
The production of recombinant proteins by transgenic
organisms is hardly new. The synthesis of commercially important proteins,
particularly pharmaceuticals, by mirco-organisms (commonly bacteria and
yeasts in bioreactors) involve processes well entrenched in industry. While
the manufacture of these proteins in high-tech bioreactors is costly, drug
manufacturers recoup investment through appropriate pricing of their products.
An alternative to the bioreactor has emerged in recent years. DNA engineering
in animals has enabled the expression of foreign proteins (commonly therapeutic
proteins) in the milk of animals such as sheep, goats and cows. These animals
become, essentially, living bioreactors that support the sustained yield
of target proteins which include human hormones, enzymes, blood coagulating
factors and immunological agents. An attraction of using transgenic animals
to produce recombinant proteins in milk lies in the lower costs of maintaining
animals as compared with brick and mortar factories. Another important consideration
is that the animals can be milked continually, thus enabling continual production
of the target protein.
Like transgenic animals, plants can also be genetically
transformed to express protein-based pharmaceuticals and other valuable
proteins. In fact, plants are even cheaper to maintain than animals. Grown
in the field, plants require little more than sunlight, water and basic
horticultural input, and as protein manufacturing factories, they are solar-powered
and ecologically friendly. Plants have yet other advantages over animals
for protein production. Their multiplication through inbred seeds is relatively
simple and efficient and they can also be propagated by vegetative means
(e.g. by cuttings, bud-grafting, etc.) without the use of seeds. In fact,
vegetative propagation serves more than just a means to multiply plants.
Since such plants are clonal copies of one another, genetically identical
copies of the best cultivars can be easily reproduced in very large numbers.
The rubber tree as a unique transgenesis model
The many advantages of transgenic plants for 'bio-pharming'
notwithstanding, their one significant weakness is the difficulty in recovering
the recombinant protein. Unlike transgenic animals where there is continual
protein production in the milk, harvesting of the recombinant protein involves
destruction of the plant or a portion of it, whether the desired protein
is to be found in the seeds, leaves or shoots. After every harvest, it takes
time for new growth to take place before the next harvest is possible. As
a result, protein recovery is more likely to be batch-wise, rather than
a continual process. Taking into consideration the strengths of the transgenic
animal (continual protein production in the milk) and the transgenic plant
(low cost of maintenance, simple clonal propagation) for recombinant protein
production, it would obviously be beneficial to have a production system
that combines both advantages. The ideal plant for recombinant protein production
would be one that is cheap to maintain and easy to multiply clonally, while
allowing for continual harvesting of the protein. This is where the transgenic
rubber tree has the distinct advantage when compared with other transgenic
crop plants.
In the bark of the rubber tree is a complex network
of laticifers, or latex vessels, each vessel merely one-third the thickness
of a human hair. These laticifers contain natural rubber latex that is exuded
when the bark is cut. Rubber tapping that is routinely practised in estates
and smallholdings is essentially the systematic and regulated cutting of
the bark to harvest the latex. Since rubber tapping is a non-destructive
method of latex extraction and harvesting, the tree can be tapped every
alternate day throughout the year without pause. Among plants, the rubber
tree is unique in its capacity to produce voluminous latex upon tapping
and to replenish this supply rapidly in readiness for the next tapping.
If Hevea brasiliensis were transformed with a gene encoding a foreign
protein, the transgenic Hevea system would allow for continual
production of the target protein, a feature lacking in any other transgenic
plant system.
In the transgenic Hevea system, therefore,
modern techniques in biotechnology meld with the generations-old practice
of rubber tapping to add new value to the rubber tree.
Inserting foreign genes into Hevea brasiliensis
The basic methods employed for genetic transformation
of the rubber tree follow procedures well-established for other plants.
As with many plants, genetic transformation of the rubber tree involves
inserting the selected gene into callus tissue (unorganised clusters of
cells) and then regenerating the transformed callus tissue into the complete
plantlet. Hevea callus tissue cultures are established from anther
walls of the rubber tree male flowers. The first transgenic Hevea
plant was produced through particle bombardment of callus tissue whereby
DNA was coated on to microscopic gold particles that were then shot into
callus tissue under high pressure. The transformed callus was subsequently
regenerated into the complete plantlet. Since this initial success, genetic
transformation has also been achieved through Agrobacterium mediation
and is today the preferred method for transforming Hevea. By this
approach, foreign genes are transferred into a bacterium called Agrobacterium
and this is then allowed to infect the callus tissue. The foreign gene is
incorporated into the genetic make-up of the Hevea callus tissue
during this process. As only a small proportion of the callus cells would
be successfully transformed, a mechanism has to be available to sort out
cells that are successfully transformed from those that are not. For this
reason, the DNA assembly that is used in transformation contains a second
gene that confers antibiotic-resistance to transformed callus cells. When
the callus tissues are transferred to culture medium containing the antibiotic,
untransformed cells perish, while the transformants - armed with the means
to resist the antibiotic - continued to thrive. The surviving callus cells
proliferate and some develop into embryo-like structures that go on to form
plantlets.
Multiplying success
From a number of transgenic plants that have been
produced, the ones that show the strongest protein expression are multiplied
for further study. Neither new nor expensive technology is needed here.
The horticultural practice of Hevea bud-grafting that is harnessed
for this purpose has its roots from the 1950s. By this approach, unlimited
clonal copies - each genetically identical - can be generated from a single
selected transformant. The amenability to clonal propagation has been proven
through successful multiplication by bud-grafting over four successive vegetative
generations of plants bearing the gus gene. Besides demonstrating
the efficiency of up-scaling transgenic Hevea, these results also
confirm the stability of the genetic transformation in this plant.
Bacterial, murine and human proteins from Hevea latex
The fact that a gene has been successfully inserted
into the rubber plant does not guarantee that the protein it encodes will
be successfully synthesised. Genes, even when they are present in the transformed
plant, can remain dormant. Another point to be watchful for is the fact
that in nature, proteins take on characteristic patterns of folding. Some
proteins become modified, for example, by having sugars linked to them.
Hence, a recombinant protein that faithfully reproduces the exact linear
sequence of amino acids of the native protein that it seeks to mimic may
still fail as a functional substitute if various structural modifications
are not in place.
In the research carried out at RRIM, transgenic rubber
plants have successfully synthesised in the latex a bacterial enzyme (beta-glucuronidase
or GUS) and a mouse antibody fragment. Significantly, these proteins are
functional proteins in that their operational characteristics are retained.
The recombinant GUS protein shows its characteristic enzymic properties
when supplied with its designated substrate, while the antibody fragment
is immuno-reactive to its matching antigenic protein. In the most recent
experiments, transgenic Hevea has produced a human protein - human
serum albumin - in its latex. Experiments with other valuable proteins are
in the pipeline.
Towards cost-efficient production of affordable proteins
Its obvious commercial potential notwithstanding,
the production of recombinant proteins from transgenic Hevea is
not about profit making alone. Cost-efficient production by transgenic plants
can alter the economics of recombinant protein synthesis. For example, hitherto
prohibitively expensive chemotherapy could be brought within reach of the
man in the street. Commercial proteins from transgenic plants need not be
confined to high-cost pharmaceuticals either. Moderate-value proteins such
as industrial enzymes or proteins used in personal care products may also
be harvested from engineered plants such as the rubber tree. In fact, the
low cost of maintaining transgenic plants make them especially suited to
high volume production of less expensive proteins that otherwise cannot
be produced cost-effectively in conventional bioreactor systems.
RRIM biotechnology makes its mark
The RRIM's transgenic research was presented at the
World Life Sciences Forum (Biovision) in Lyon at the beginning of the year.
This project has been featured in the news media, both local and foreign.
It has appeared on Malaysian television and in the syndicated Discovery
television programme. Articles have appeared in Malaysian newspapers such
as The Star, The New Straits Times, Berita Harian
and Sing Chew Jit Poh, and in the foreign press, including Newsday
in the United States and The Times, The Observer and New
Scientist in the United Kingdom. The project has won awards at the
MINDEX-INNOTEX exhibition in Kuala Lumpur, the Salon International
des Inventions in Geneva and the INPEX exhibition in Pittsburg.
Advantages of the transgenic rubber tree as a living protein
factory
There are several advantages in using transgenic
Hevea for the production of commercially valuable proteins. Among
these are:
- The concept is a novel approach to cost-efficient
production of high value proteins in the latex of transgenic rubber
trees, which essentially serve as production lines.
- The approach is environment-friendly. The process
is driven by the sun and is therefore energy-efficient and essentially
pollution-free.
- Rubber trees require no special attention beyond
routine horticultural maintenance. Their use is thus highly cost-efficient
as compared with conventional bioreactor systems.
- Production of the target protein is continual through
a system of non-destructive harvesting (tapping) of the rubber tree.
- Glycosylation of eukaryote proteins (binding of sugars
to certain proteins to render them functional), which does not occur
in bacterial systems of protein production, can take place in the transgenic
rubber tree.
- The latex that exudes from the rubber tree is free
of animal viruses and other contagion vectors. These include pathogenic
viruses such as those causing AIDS or hepatitis, and prions that cause
mad cow disease and its human variant.
- Successful transformation of the rubber tree for
a specific gene needs to be achieved only once. Rubber trees are amenable
to vegetative propagation and an unlimited number of genetically identical
plants (clones) can be generated by conventional horticultural methods.
- The methodology does not involve the use of animals
and hence the issue of animal rights does not arise.
- From the biosafety viewpoint, the transgenic rubber
tree raises far fewer objections as compared with other crops. Hevea
is not native to Malaysia and propagation is normally by vegetative
means. Hence, it is not expected to have adverse effect on the environment
or on the crop. Unlike transgenic food products, recombinant proteins
from Hevea are purified from the transgenic elements that are
not presented to the consumer.
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