Download 2-3. Gene technology

Recombinant gene technology
Content:
1. Biotechnology, gene technology, society
2. Genetic recombination
3. Gene delivery to cultured cells
4. Genetically Modified Organisms (GMOs)
4a. Transgenic animals in basic research
4b. Knock-out animals
4c. In vivo RNA interference
4d. Genetically Modified Organisms (GMOs) in the economy
4e. Genetically modified microorganisms
5. Recombinant proteins
6. Antibody technology
7. Antibiotics
1. Biotechnology, gene technology, society
SLIDE 1, 2 Gene technology is the alteration of DNA by means of molecular genetic methods. In other
words, gene technology is a set of tools, used for altering the genetic material generating recombinant DNAs,
recombinant cells or recombinant animals. Gene technology can be opposed to genomics, which investigates
the structure and function of the genetic material, but does not alter it.
SLIDE 3 EXTRA REQUIREMENT Gene technology – top danger Naturally, gene technology met
disapproval by a significant part of the society. Interestingly, initial doubts were raised by the scientists
themselves.
SLIDE 4 EXTRA REQUIREMENT Moratorium, then Asilomar conference The Asilomar Conference
on Recombinant DNA was an influential conference organized by Paul Berg discussing the potential
biohazards and regulation of biotechnology held in February 1975 at a conference center Asilomar. A group of
around 140 professionals (primarily biologists, but also lawyers and physicians) participated in the conference
to draw up voluntary guidelines to ensure the safety of recombinant DNA technology. The conference also
placed scientific research more into the public domain, and can be seen as applying a version of the
precautionary principle. The repercussions of these actions are still being felt through the biotechnology
industry and the participation of the general public in scientific discourse. Due to potential safety hazards,
scientists worldwide had halted experiments using recombinant DNA technology, which entailed combining
DNAs from different organisms. After the establishment of the guidelines during the conference, scientists
continued with their research, which increased fundamental knowledge about biology and the public’s interest
in biomedical research.
SLIDE 5 EXTRA REQUIREMENT Today’s worries
1. Human-made contagious diseases - AIDS from poliomyelitis vaccine?
2. We alter human evolution
3. We interfere to the Creator’s business
4. Genetically modified foods are unhealthy
5. We change the ecosystems
1
2. Genetic recombination
SLIDE 6 Genetic recombination As we shown, DNA fragments can be inserted to plasmids (and also to
viruses with small genome size) by means of RE/ligase system. However, foreign genes cannot be inserted to
large genomes with this technique because large DNAs are full of restriction recognition sites, therefore RE
digestion would result in a large number of fragments. Therefore, we use genetic recombination for the
insertion of exogenous DNA to large genomes. If the foreign DNA randomly integrates to the genome, we talk
about illegitimate („non legal”, random) recombination, or if, it is integrated by a recombination mediated by
homologous sequences, we talk about homologous recombination.
SLIDE 7 Illegitimate recombination generally results in the integration of foreign DNA in multiple copies
located close to each other in a tandem orientation.
SLIDE 8 Homologous recombination is a DNA exchange between two identical DNA sequences. For
achieving homologous recombination, we first isolate the DNA sequence from the genome, which is intended
to use as the site for foreign gene insertion. Subsequently, the isolated DNA fragment is inserted to a plasmid,
followed by the insertion of foreign gene to the subcloned genomic DNA. The two arms of the cloned genomic
DNA will subserve as homologous sequences for the recombination. As a result of homologous recombination,
the foreign DNA sequence will be inserted to the genome. Thus, in contrast to illegitimate recombination
resulting in a random DNA integration, homologous recombination results in targeted gene insertion.
3. Gene delivery to cultured cells
SLIDE 9 Cultured cell types We can culture various types of cells in vitro. (1) Normal body cells (such as
fibroblast cells) divide in vitro as many times as they do in the body. These cells are called primary cell culture.
Primary culture can be produced using post-mitotic cells (neurons and myocardiocytes), too. (2) Tumor cell
lines (isolated from tumors) have the capability for unlimited propagation. (3) The so called immortalized cell
lines are also capable for unlimited proliferation, but they retain the characteristics of the original cell type,
and form a monolayer culture in a Petri dish.
SLIDE 10, 11 Gene delivery techniques to cultured cells Foreign genes can be delivered by
physico/chemical or viral-based methods. The most frequently used non-viral method is based on liposomes.
Liposomes are spherical structures composed of phospholipid bilayers. They can carry DNA and other drugs
(both hydrophilic and hydrophobic materials). Materials carried by the liposomes get to the cytoplasm by the
fusion of phospholipid membranes of liposomes and cells. The most frequently used viruses for gene delivery
are as follows: adenoviruses (do not integrate to the genome), retroviruses and adeno-associated viruses (both
of them integrate to the genome).
SLIDE 12 Foreign gene transfer – transgene For the sake of simplicity, consider a transgene encoding a
protein, which produce blue color in transduced cells, e.g. β-galactosidase (encoded by the lacZ gene) in the
presence of X-Gal substrate. It can be seen that gene delivery has a certain efficiency, which can be up to 50%
in immortalized cells, but very low (1%) in primary cells by using chemical methods. Virus-based gene delivery
can reach 100% in primary cell culture.
SLIDE 13 Foreign gene – transient expression If a foreign gene (equipped with the appropriate
regulatory sequences) does not integrate to the host chromosome, it is normally expressed transiently (for a
short time) for which the reason is that non-integrated DNAs are eliminated by the cells.
SLIDE 14 Foreign gene – stabile expression If a foreign gene is inserted to the host genome, it is
considered hereafter as own DNA, and the expression will be stable (long-term) provided that the foreign gene
is integrated to a permissive (opposite of repressive) genomic environment. Repressive genomic environment
is for example the heterochromatin regions (telomers and centromer regions) of the chromosomes, which are
heavily methylated both at the histone and DNA levels.
4. Genetically Modified Organisms (GMOs)
SLIDE 15 Genetically modified organisms (GMOs; Genetically Modified Organisms) harbor one or more
foreign genes (derived from other species. We can group GMOs according to the aim of their generations or their
taxonomy. According to the aim of generation (1) we can used them in basic science, that is for gaining
information having no direct economic relevance, or (2) we can apply them in medicine, agriculture or industry.
According to the taxonomic classification GMOs can be microorganisms, plants, animals and human.
2
4a. Transgenic animals in basic research
SLIDE 16 A transgenic animals contain one or more foreign genes in their genomes. Exogenous genes are
present in all of the cells of the animals, but are not necessarily expressed in all of the cells. Transgenic
animals can be used as (1) models for the investigation of the function or the cellular effect of the inserted gene,
or (2) they can be economically important livestock.
SLIDE 17 Gene delivery to living organisms can be classified on the basis whether the foreign gene is
present in every cell of the body (germ line gene delivery); or only in specific cells (somatic gene delivery). (1)
Gene delivery to germ line cells We can used natural germ line cells (fertilized egg cells) or e.g. pluripotent ES
cells for generation transgenic organisms. Foreign genes can be delivered to the male pronucleus of zygote by
microinjection. Foreign genes can also be delivered by retrovirus vectors. Alternatively, ES cells can be used for
gene delivery to the whole organism. (2) Gene delivery to body (somatic) cells is normally performed in adult
individuals. Currently, virus vector-based approaches are far the most efficient techniques for the delivery of
foreign genes to the adult body. Gene delivery to the target tissue can be performed directly; or in a cell-based
manner. In this latter technique, foreign genes are first delivered to stem cells, followed by the implantation of
transformed cells to the body. Liposomes are often supplied by a protective layer in in vivo applications and by
homing peptides which target the liposomes to specific cells. Homing peptide can be for example, a peptide
ligand of a receptor, which directs liposomes to the cells harboring this receptor.
SLIDE 18 Transgenic animals: foreign gene delivery to the zygote by pronuclear microinjection
Foreign genes are equipped by regulatory sequences. DNA is transferred to the male pronucleus of the zygote.
In the mean time, the surrogate mother (to whom the genetically manipulated zygote is transplanted) is
treated by hormones and copulated with a sterile male. Progenies are analyzed for the presence of the
transgene by PCR using their tails as the source of DNA. Founders are those mice which harbor the transgene
in their genomes, and are further bred to obtain homozygous lines.
SLIDE 19 Transgenic animals: foreign gene delivery retrovirus vector The protocol is similar as
described above, except that instead of microinjection, we infect zygotes by retroviruses.
SLIDE 20, 21 EXTRA REQUIREMENT Transgenic animals: foreign gene delivery to ES cells The
following protocol results in the generation of transgenic animals in ES cell model. (1) Isolation of blastocyst
from the donor female; (2) preparation of ES cells from the inner cell mass; (3) delivery of a recombinant
plasmid containing the foreign gene and an antibiotics resistance gene to ES cells; (4) transformed cells are
selected on the basis of antibiotic resistance (non-transformed cells die as a result of cytotoxic effect of
antibiotics); (5) generation of another blastocyst by new donor parents, (6) which are used for the inoculation
of transformed cells, followed by (7) the transfer of modified blastocyst to the womb of recipient females. (8)
Offspring will be genetically mosaics, since genetically modified cells represent only a certain proportion of
cells of the inner cell mass. (9) Offspring harboring the foreign gene in their germ line cells are bred with
normal mice thereby producing heterozygote offspring, which subsequently (10) are inbred (11) to obtain
homozygote animals (containing the transgenes on both homologous chromosomes).
SLIDE 22 EXTRA REQUIREMENT Regulation of gene expression If a foreign gene is present in every
cell of an organism, its expression should be restricted to specific cells. Expression of the transgene can have a
disturbing effect or can even be toxic in non-desired cells. We talk about constitutive gene expression when the
transgene is continuously expressed. The transgene can be expressed in (1) every cell (ubiquitous expression);
(2) every similar cell type (e.g. in each type of neuron); or only (3) certain cell types (e.g. in pyramidal cells of
hippocampus). There are technical possibilities to induce or repress the expression of foreign genes. Foreign
genes cause gain-of-function phenotypes, while knocking out a gene causes loss-of-function phenotype.
Knocking out a gene causes null expression. RNA interference blocks the expression of transcribed mRNAs
therefore, it is called post-transcriptional regulation. By means of RNAi, we can control gene expression in
adult individuals, which offers a great perspective in medicine. The major obstacle in the utilization of this
technique is the targeting of the double-stranded RNA-s to the desired cells.
SLIDE 23 EXTRA REQUIREMENT Cell-specific gene expression The expression of a particular
transgene is usually desired in only specific cells. The problems in solving this task are that the regulatory
elements of eukaryotic genes are dispersed in a very long DNA stretch, and that we do not know which are
important in the faithful expression of the particular gene. These problems can be solved by the so called BAC
(bacterial artificial chromosome) technology. The BAC is a low copy number bacterial plasmid (such as the F
plasmid), which can harbor large DNA segments. Transgene is inserted to the BAC in place of a gene, which is
expressed in the desired cell, so the transgene will have the same regulatory elements as the original gene had.
3
The BAC clone is inserted to the genome by illegitimate recombination, thus the original gene will not be
knocked out. This technique results in similar expression of the transgene to that of the gene whose regulatory
elements was utilized.
4b. Knock-out animals
SLIDE 24 Knockout animals – Nobel Prize The development of knockout technology was awarded by a
Nobel Prize in 2007 to Martin Evans for his work on embryonic stem cells, and Mario Capecchi and Oliver
Smithies for the development of homologous recombination technique in a mouse model. Homologous
recombination can also be used for the replacement of malfunctioned genes with intact ones, which has a great
significance in medicine.
SLIDE 25 EXTRA REQUIREMENT Generation of knockout animals Generally, a transgene is
inserted to an arbitrary part of the genome by means of illegitimate recombination. However, if we want to
delete a gene, it can be carried out in a targeted way using homologous recombination. This technique is based
on a dual selection system. A neomycin phosphotransferase (neo; confers resistance against the neomycin
antibiotics) gene is inserted within the subcloned DNA fragments, which serves homologous arms for the
recombination to the desired site of the genome resulting in the deletion of targeted gene. This is a positive
selection system since only those cells survive neomycin treatments, which harbors the neo genes. The
problem is that the chance for the illegitimate recombination of a DNA fragment to the genome is
approximately 100 fold higher than the frequency of homologous recombination. It means that in the majority
of neo+ cells the targeted gene is not deleted. In order to circumvent this problem, a negative selection gene,
the herpes simplex virus thymidine kinase gene; (tk) is also included in the targeting plasmid. The tk gene is
located outside of the homologous flanking sequences; therefore, in contrast to illegitimate recombination,
upon homologous recombination tk gene will not integrate to the genome. The substrate of TK enzyme is the
acyclovir, which if phophorylated by the TK, becomes toxic to the cells. So, this dual selection system operates
as follows. The targeting plasmid (see slide 27) is transferred to the ES cells. At the first round of selection
every neo+ cells will be selected (non-transformant cells will die as a result of antibiotics treatment. After the
first selection we have two cell populations; (1) the majority of transformed cells harbor the recombinant
plasmid integrated by illegitimate recombination, therefore containing both neo and tk; (2) about 1% of cells
containing only neo gene because homologous recombination excluded the tk gene. Thus, application of
acyclovir selection against tk+ cells will result in the survival of neo+ but tk- transformed cells. In other words,
with the utilization of this dual selection system, we obtain cells with the deleted gene.
SLIDE 26 EXTRA REQUIREMENT Generation of knockout animals in ES cells The protocol of the
generation of knockout animals is very similar to the above ES cell-based transgenic technique. The major
differences are as follows: (1) the presence of negative selection marker (tk gene) in the targeting plasmid, and
(2) that transgenesis results in gain of function, while targeted gene deletion (knockout technique) results in
loss-of-function. The targeted gene is inactivated in only one of the two homologous chromosomes; therefore
inbreeding of genetically modified animals is needed to generate knockout animals lacking both copies of the
given gene.
6. in vivo RNA interference
SLIDE 27-29 EXTRA REQUIREMENT RNA interference Silencing of the activity of specific genes is
very important in both basic research and medicine. RNA interference (RNAi) is far the most effective tool for
this purpose. RNAi performs very well in cell culture and in primitive organisms, such as C. elegans and fruit
fly. The major obstacle for using this technique in human medicine is the ineffectiveness of techniques for
targeting RNAi-mediating nucleic acids to the desired cells. RNAi can be used in medicine to block the
expression of malfunctioned genes (e.g. in tumor cells). RNAi can be evoked by the following nucleic acids: (1)
long double-stranded RNA molecules (they cannot be used in mammals, because cells interpret them as
viruses and, therefore apoptosis is triggered); (2) siRNAs (si: small interfering); or (3) shRNA-coding DNAs
(sh: small hairpin). shRNAs are converted to siRNAs by the DICER. siRNAs evoke RNAi, which results in the
inactivation of the target mRNAs. The shRNA-encoding DNAs can be delivered by (3a) physico/chemical
methods or by (3b) viral vectors. Lentiviruses (belonging to retroviruses) integrate to the host cell genome,
while adenoviruses remain extrachromosomal, therefore, expressing shRNAs transiently (for a short time).
RNAi-mediating nucleic acids can be delivered locally or systemically. RNAi takes place in the P body of the
cell. P body contains many enzymes involved in mRNA turnover.
4
4d. Genetically Modified Organisms in the economy
SLIDE 30 EXTRA REQUIREMENT GMO – Society protest People do not really like GMOs, especially
genetically modified foods. A ususal claim is that they can be unhealthy. This argument is frequently used by EU
authorities against importing American agricultural products (economical protectionism).
SLIDE 31-33 Genetically modified plants Until now, tomatoes have been harvested while they are still green,
to avoid them ripening and going soft before they reach the market. They are then treated with ethylene gas to
turn them red but they do not really develop any flavor. The first genetically modified agricultural plant was the
tomato (phantasy name: Flavr-Savr, from flavour savour). It was approved by the FDA (Federal Drug
Administration; this office allows, among others, the GMOs to get to the market). Flavr Savr was a failure.
Opinions differ about the reason: some people say that consumers were not prepared for a genetically modified
food, other claim that the choice of the breed was not good (in was not delicious). A major problem of tomato
trade is that tomato ripe very soon, and therefore it cannot be stored for a prolonged time in the stores. Calgene, a
biotech company solved this problem as follows. They introduced the antisense RNA of PG gene (which is
responsible for the ripening) to the tomato, thereby inhibited the production PG protein. At this time they did not
know about a more efficient system, called RNA interference. Genetically modified crops
First generation GMOs (the producers benefit): The genome of the maize was modified to contain herbicideor pesticide-resistance genes, or the gene of Bacillus thuringiensis, which provides protection against insects. The
genomes of soybean, cotton, rape, sugar cane and sugar-beet were modified to express pesticides or herbicides.
Second generation GMOs (the consumers benefit): oil content in soybean and rape was modified; fusarium
resistant wheat, ferritin containing lettuce, etc. Rice contains a few amount of vitamin A, which is a problem in
countries where rice is the sole food. In the frame of „Golden Rice” project foreign genes playing roles in the
synthesis of β-carotene (precursor of vitamin A) were inserted.
Third generation GMOs (companies benefit) can be considered as bioreactors, or factories, because the
produce materials, which are normally produced by the industry: vaccines, lactoferrine, hormones, clotting factors,
etc.
SLIDE 33b EXTRA REQUIREMENT Terminator and traitor crops An often cited controversy is a
"Technology Protection" technology dubbed 'Terminator'. This yet-to-be-commercialized technology would
allow the production of first generation crops that would not generate seeds in the second generation because the
plants yield sterile seeds. The patent for this so-called "terminator" gene technology is owned by Monsanto
Company. In addition to the commercial protection of proprietary technology in self-pollinating crops such as
soybean (a generally contentious issue), another purpose of the terminator gene is to prevent the escape of
genetically modified traits from cross-pollinating crops into wild-type species by sterilizing any resultant hybrids.
Ironically, the terminator gene technology created a backlash among the same groups that considered outcrossing
of GM plants dangerous. They felt the technology would prevent re-use of seed by farmers growing such
terminator varieties in the developing world and was ostensibly a means to exercise patent claims. Similarly, the
hypothetical Trait-specific Genetic Use Restriction Technology, also known as 'Traitor' requires application of a
chemical to genetically modified crops to reactivate engineered traits. This technology is intended both to limit the
spread of genetically engineered plants, and to require farmers to pay yearly to reactivate the genetically
engineered traits of their crops. Traitor is under development by companies including Monsanto. Hybrid seeds
were commonly used in the developed countries long before the introduction of GM crops. Hybrid seeds cannot
be saved, so purchasing new seed every year is already a standard agricultural practice. MON810 (maze
containing Bt toxin) is the only GMO crop cultivated in European soil. However, in addition to France and
Germany, other European countries that have placed bans on the cultivation and sale of GMOs include Austria,
Hungary, Greece and Luxemburg. Ireland has also banned GMO cultivation, and has instituted a voluntary label
for GMO-free food products. Poland has also has tried to institute a ban, with backlash from the European
Commission Now Bulgaria is pushing for a 5-year ban.
SLIDE 34 EXTRA REQUIREMENT How to modify plants using genetic techniques? Let’s see how a
genetically modified crop can be generated, using the example of cold-tolerant tomato. First, we explain some
important information. The differentiated cells of tomato can be very easily made totipotent in in vitro conditions.
Agrobacterium tumefaciens is the most popular gene delivery vector for plants. This bacterium is propagated in
plants in such a way that it induces tumors on the plant and consumes the fresh tumor cells. The bacterium
induces tumor formation by means of its Ti plasmid. To be more precise, a small piece (called T-DNA) detaches
from the Ti plasmid, and integrates, to the genome of plant cells. Investigators modified T-DNA in such a way that
it retained its ability to integrate into the plant genome, but lost its tumor forming capability. Various foreign
DNAs can be inserted to the T-DNA. In the case of cold-tolerant tomato the anti-freeze protein of a see fish was
introduced to the tomato genome (equipped with plant regulatory elements, of course). This gene was delivered to
the tomato genome using the Ti plasmid as a vector. The product of anti-freeze gene is an enzyme, which plays a
5
role in the synthesis of saturated fatty acids of cell membrane. The higher the number of saturated phospholipids
in the membrane the, higher is the viscosity of the membrane, that is, the resistance against cold is higher. Ti
plasmid contains an antibiotic resistance cassette, too. Tomato’s leaves were placed onto antibiotics-containing
culture medium, and then they were infected with recombinant Ti-plasmid containing Agrobacterium. Antibiotics
kill cells without antibiotic resistance gene, only transformant cells (containing antibiotic resistance and antifreeze gene) survive. Finally, we regenerate tomato. Each of the cells of these plants contained the anti-freeze gene.
SLIDE 35 A 3rd generation GM animal Let’s consider a general scheme of the production of a genetically
modified animal. The product of human ATIII gene play a role in the resolvation of clots =coagulates) formed in
the blood stream, that is ATIII has a thrombolytic effect. Some people produce too much clots, therefore the risk
of heart attack is high in these persons. It is advisable to overproduce ATIII for a prevention purpose. ATIII gene
is introduced to the genome of a goat in the form o fan expression cassette equipped with a promoter/enhancer
allows expression the cell secreting to the milk of the goat (cell-specific gene expression). The result: ATIII protein
in the goat milk, which has to be purified and administer intravenously.
SLIDE 36 Genetically modified animals Various transgenic projects are in progress. For example, a rapidly
growing salmon, enhanced level of caseins in cow milk, human-compatible pig’s liver.
SLIDE 37 EXTRA REQUIREMENT Genetically modified human There could be two reason for the
generation of transgenic humans. (1) germ line gene therapy: correcting genetic diseases; (2) cosmetics-aimed
gene delivery.
4e. Genetically modified microorganisms
SLIDE 38 Microorganisms viruses, bacteria and unicellular fungi belong to this category.
Viruses are applied in many fields of basic and applied research and medicine. Viruses had millions of years to
develop effective tools for invading cells. Scientists utilize this feature in basic research and gene therapy, in such
a way that the cytotoxicity of the virus is eliminated or minimized, while the ability to penetration to cells is
retained or even improved. Viruses are also used tumor therapy and in brain research for labeling neural circuits.
Recombinant viruses are also used as live vaccines for immunization against themselves or against other
pathogens (for that latter possibility, a part of the DNA of this pathogen has to be inserted to the virus genome).
Bacteria: Escherichia coli is one of the most important model organism of molecular biology. In biotechnology,
several bacterial species are used in various fermentations processes. Gene technology is used to generate bacteria
to produce recombinant proteins
Unicellular fungi: A large sort of proteins and other organism materials are produced by fermentation
technologies using various fungi species.
5. Recombinant proteins
SLIDE 39 Genetically modified bacteria are used for the production of recombinant proteins, which are
important medicine. Here are some examples:
Recombinant proteins
Insulin
Clotting factors
Growth hormones:
Disease
Diabetes
hemophilia
dwarfism
Advantage: they cannot cause illness (in contrast to the proteins obtained earlier from corps) Disadvantage:
proteins are processed in a different way in bacteria (e.g. there is no glycosylation in bacteria)
SLIDE 40 Bacterium DNA Foreign genes can be inserted both to the genomial DNA of bacteria or to their
plasmids. Plasmids can be integrating (to the genome, such as F factor), or non-integrating. Plasmids can be large
sized with low copy number, or small sized, with high copy number.
SLIDE 41 Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1) which in
turn starts many protein activation cascades (2). These include: translocation of glucose transporter to the plasma
membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and fatty acid syntheses synthesis (6).
SLIDE 42 Recombinant insulin Diabetes is one of the top three causes of death in the industrial world.
Type I diabetes sets in before the 20th year and is caused by autoimmune destruction of the insulin-forming
cells in the pancreas. Type II diabetes, by contrast, tends to develop in middle age (91% of all diabetics),
especially in overweigh people. Millions (171 million) of diabetics were treated with insulin every day. The
6
insulin was obtained from the pancreases of pigs and cattle. One single diabetic patient would require the
pancreas of 50 pigs to cover a year’s supply. Hoechst, one of the major insulin suppliers at the time, processed
eleven tons of pigs’ pancreas per day, provided by over 100,000 slaughtered animals. In July 1980, 17
volunteers were given insulin injections that made the headlines in the newspaper. Today, insulin is produced
by genetically transformed fungal cells using fermentation.
SLIDE 43 EXTRA REQUIREMENT Protein engineering is the alteration of protein structre by using
methods of molecular genetics. Two main approaches are used in modern protein engineering: (1) Directed
Evolution approach, which is based on the random generation of multiple mutations in a gene, followed by
screening for desirable changes in the properties of the protein. This technique in protein engineering can be
described as the man-made version of natural selection. In this process, the protein engineering team generates a
library of variants of the target protein by generating mutations. This library is then screened for variants showing
the desired properties. One of the improved variants is taken as the basis of a new cycle of the mutagenesis,
screening and selection. This process of variant generation, screening and selection is repeated until a variant of
the protein has been identified that fulfills the criteria for the successful use of the enzyme. (2) The alternative
approach is called Rational Protein Engineering, which uses knowledge-based approaches, i.e., scientists
determine the 3D structure of the enzyme (with X-ray crystallography or NMR technique). The 3D structure sheds
light on the function of the various amino acids of a protein. This information is used to produce targeted
mutations in the enzyme. In practice, many protein engineering projects rely on a combination of both approaches,
as they complement each other.
11. Antibody technology SLIDE 44-49
Polyclonal antibodies The classical method of obtaining antibodies is the immunization of laboratory
animals, by injecting them with an antigen. The immunization process must be repeated several times
successfully before antibodies can be isolated from the animal’s sera. The obtained antibodies are a mixture of
molecules that bind to various sites (epitopes) of the antigen. The strength of these bonds varies. Each of these
specific antibodies is produced by its own B-lymphocyte clone in the blood.
Monoclonal antibodies The method developed by Köhler and Milstein uses the hybridoma technique. In a
first step, the lab animal (usually a mouse) is inoculated with an antigen, and the antibodies to the antigen are
first produced in the spleen, then circulate in the blood and lymph system. These are a range of antibodies,
derived from various cell clones, i.e., polyclonal antibodies. The antibodies are not taken from the blood but
from the spleen of the inoculated mouse. The large number of B-lymphocytes can be easily isolated. Blymphocytes originate from bone marrow stem cells and reproduce in the spleen and lymph node, producing
antigen-specific clones or differentiating into plasma cells. The B-lymphocytes undergo in vitro fusion with
myeloma cells, which are tumor cells that are easily cultured. The resulting hybridoma cells give rise to clones
that produce uniform antibodies (monoclonal antibodies). The selected clones have the immortality of cancer
cells, combined with the antibody production of lymphocytes and are therefore ready to be produced in
unlimited quantities.
Recombinant antibodies A third alternative is recombinant antibodies that are not produced in animals (in
vivo) but in bacterial or eukaryotic cell culture (in vitro).
12. Antibiotics
SLIDE 50, 51 In 1928, Alexander Fleming made one of the most important contributions to the field of
antibiotics. In an experiment, he found that a strain of green Penicillium mold inhibited the growth of bacteria
on an agar plate. This led to the development of the first modern era antibiotic, penicillin. A few years later in
1932, a paper was published which suggested a method for treating infected wounds using a penicillin
preparation. Although these early samples of penicillin were functional, they were not reliable and further
refinements were needed. These improvements came in the early 1940s when Howard Florey and associates
discovered a new strain of Penicillium, which produced high yields of penicillin. This allowed large-scale
production of penicillin, which helped launch the modern antibiotics industry. After the discovery of penicillin,
other antibiotics were sought. In 1939, work began on the isolation of potential antibiotic products from the
soil bacteria streptomyces. It was around this time that the term antibiotic was introduced. Selman Waxman
and associates discovered streptomycin in 1944. Subsequent studies resulted in the discovery of a host of new,
different antibiotics including actinomycin and neomycin all produced by Streptomyces. Other antibiotics that
have been discovered since include bacitracin, chloramphenicol and tetracyclines. Since the 1970s, most new
antibiotics have been synthetic modifications of naturally occurring antibiotics. Although most antibiotics
7
occur in nature, they are not normally available in the quantities necessary for large-scale production. For this
reason, a fermentation process was developed. It involves isolating a desired microorganism, fueling growth of
the culture and refining and isolating the final antibiotic product. It is important that sterile conditions be
maintained throughout the manufacturing process, because contamination by foreign microbes will ruin the
fermentation. Microorganisms used in fermentation are rarely identical to the wild type. This is because
species are often genetically modified to yield the maximum amounts of antibiotics. Mutation is often used,
and is encouraged by introducing mutations such as ultraviolet radiation, x-rays or certain chemicals.
Selection and further reproduction of the higher yielding strains over many generations can raise yields by 20fold or more. Another technique used to increase yields is gene amplification, where copies of genes coding for
enzymes involved in the antibiotic production can be inserted back into a cell, via vectors such as plasmids.
8