Role Of Genetic Engineering In Improvement Of Pharmaceutical Production of Microorganisms lecture in department of biology.faculty of science.University of Kufa

1. Role Of Genetic Engineering
In Improvement Of
Pharmaceutical Production
of Microorganisms
Dr.Nawfal Hussein Aldujaili
Department of biology ,College of Science
University of Kufa, 24 March , 2011
Conference Marcht 24, 2011

2. Strain Improvement
 - After an organism producing a valuable product is
identified, it becomes necessary to increase the
product yield from fermentation to minimise
production costs. Product yields can be increased by
(i) developing a suitable medium for fermentation,
(ii) refining the fermentation process and
(iii) improving the productivity of the strain.

3. Strain Improvement
 The techniques and approaches used to genetically
modify strains, to increase the production of the desired
product are called strain improvement or strain
development.
Strain improvement is based on the following three
approaches:
(i) mutant selection,
(ii) recombination, and
(iii) recombinant DNA technology.

4. Strain improvement
 Virtually all biotherapeutic agents in clinical use are
biotech pharmaceuticals.
A biotech pharmaceutical is
 simply any medically useful drug whose manufacture
involves microorganisms or substances that living
organisms produce (e.g., enzymes).
 Most biotech pharmaceuticals are recombinant—that is,
produced by genetic engineering. Insulin was among the
earliest recombinant drugs.

5. Recombinant DNA Technology
 The ability to
combine the DNA of
one organism with
the DNA of another
organism.
 Recombinant DNA
technology was first
used in the 1970’s
with bacteria.
1. Remove bacterial DNA
(plasmid).
2. Cut the Bacterial DNA with
“restriction enzymes”.
3. Cut the DNA from another
organism with “restriction
enzymes”.
4. Combine the cut pieces of
DNA together with another
enzyme and insert them into
bacteria.
5. Reproduce the recombinant
bacteria.
6. The foreign genes will be
expressed in the bacteria.

7.  Bacterial Transformation
• The ability of bacteria to
take in DNA from their
surrounding environment
• Bacteria must be made
competent to take up
DNA
Microorganisms as Tools

8. Yeast are Important Too!
 Single celled eukaryote
 Kingdom: Fungi
 Over 1.5 million species
 Source of antibiotics, blood cholesterol lowering
drugs
 Able to do post translational modifications
 Grow anaerobic or aerobic
 Examples: Pichia pastoris (grows to a higher
density than most laboratory strains), has a no.
of strong promoters, can be used in batch
processes

9.  Cloning and Expression Techniques
• Fusion Proteins
Microorganisms as Tools

10.  Yeast Two-Hybrid System
• Used to study protein interactions
Microorganisms as Tools

11. Recombinant Microorganisms
 The revolutionary exploitation of microbial genetic discoveries in the
1970s, 1980s and 1990s depended heavily upon the solid structure of
industrial microbiology, described above.
 The major microbial hosts for production of recombinant proteins are
E. coli, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula
polymorpha and Aspergillus niger.
 The use of recombinant microorganisms provided the techniques and
experience necessary for the successful application of higher
organisms, such as mammalian and insect cell culture, and transgenic
animals and plants as hosts for the production of glycosylated
recombinant proteins.

13. 13
The Production of Commercial Products by
Recombinants Microorganisms
 Molecular biotechnology can be used to enhance
the production of many commercially important
compounds e.g.
 Vitamins
 Amino acids
 Antibiotics
 We will be investigating the use of recombinant
organisms to improve or enhance the production
of :
 Restriction enzymes
 Ascorbic acid
 Microbial synthesis of the dye indigo
 Production of xanthan gum

14. 14
Therapeutic Agents
 Before the advent of molecular biotechnology
most human proteins were available in only
small (limited) quantities.
 Today hundreds of genes for human proteins
have been cloned, sequenced, expressed in the
host cells and are being tested as therapeutic
agents (drugs) in humans.

15. Types of biomolecules produced through
recombinant DNA technology
 Recombinant Hormones
 Insulin (and its analogs), growth hormone, follicle stimulating hormone,
salmon calcitonin.
 Blood products
 Albumin, thrombolytics, fibrinolytics, and clotting factors ( Factor VII, Factor
IX, tissue plasminogen activator, recombinant hirudin )
 Cytokines and growth factors
 Interferons, interleukins and colony stimulating factors (Interferon, α, β and
γ, erythropoietin, interlukin-2, GM-CSF, GCSF )
 Monoclonal antibodies and related products
 Mouse, chimeric or humanized; whole molecule or fragment; single chain or
bispecific; and conjugated (rituximab, trastuzmab, infliximab, bevacizumab)

16.  Recombinant Vaccines
 Recombinant protein or peptides, DNA plasmid and anti-idiotype
(HBsAg vaccine, HPV vaccine)
 Recombinant Enzymes
 Dornase– α (Pulomozyme), Acid glucosidase (Myozyme), α –L-
iduronidase (Aldurazyme) and Urate Oxidase
 Miscellaneous products
 Bone morphogenic protein, conjugate antibody, pegylated
recombinant proteins, antagonist

18.  The market for recombinant therapeutics has
considerably improved with generation of new molecules
and new expression systems. Even though the overall
world market has been around $30-40 billion it is
expected to reach around $75 billion by end of this
decade.
 In fact the major money producer has been few
biomolecules such as insulin, erythropoietin, interferon
and hormones. These few molecules take a major share
of biopharmaceutical market (Table 2). Among these
biomolecules, erythropoietin is the most valuable product
followed by insulin, growth factors and interleukin. It is
projected that erythropoietin market will be around $10
billion by next five years.

19. Human therapeutics from recombinant DNA
technology
 One of the greatest benefit of the recombinant DNA technology has been the
production of human therapeutics such as hormones, growth factors and antibodies
which are not only scarcely available but also are very costly for human use. Ever
since the recombinant insulin was produced by Eli Lilly in 1982, considerable efforts
has been made world wide to clone and express many therapeutically important
proteins, which are otherwise difficult to produce either by extraction from the natural
sources or by chemical synthesis.
 Therapeutic proteins are preferred over conventional drugs because of their higher
specificity and absence of side effects. Therapeutic proteins are less toxic than
chemical drugs and are neither carcinogenic nor teratogenic. Further, once the
biologically active form of a protein is identified for medical application, its further
development into a medicinal product involves fewer risks than chemical drugs.
Notable diseases for which recombinant therapeutics have been produced include
diabetes, hemophilia, hepatitis, myocardial infarction and various cancers.
 Recombinant therapeutics include proteins that help the body to fight infection or to
carry out specific functions such as blood factors, hormones, growth factors,
interferons and interleukins. Starting with simple protein such as insulin and then
growth hormone, recombinant biopharmaceuticals has increased considerably in
recent years.

20.  Till today, around 165 biopharmaceuticals (recombinant proteins,
antibodies, and nucleic acid based drugs) have been approved.
Table 1 lists the type of biomolecules that have been produced by
the recombinant DNA technology. This includes hormones, growth
factors, blood products, monoclonal antibody, enzymes and many
others.
 Production using recombinant DNA technology has made these
molecules available for the treatment of human diseases at a
relatively lower cost. Availability of large amount of pure molecules
has helped in development of its different modified form to have
improved pharmacokinetic parameters.
 Pegylated proteins and controlled release formulation of
biomolecules have become reality with improved characteristics.
This has not only helped in cheap availability of the biomolecules for
health care but also has led to development of new molecules
having improved performances. The best example being different
varieties of insulin analogs (long acting , slow release, acid stable
etc). Others are being pegylated proteins such as peg-interferon
and peg-antibodies and growth factors.

21. The most notable applications of the recombinant
technology having direct impact on humanity have been:
 1. Large scale production of therapeutic protein such as insulin,
hormones, vaccine and interleukins using recombinant
microorganisms.
 2. Production of humanized monoclonal antibodies for therapeutic
application
 3. Production of insect resistant cotton plant by incorporation of
insecticidal toxin of Bacillus thuringiensis (Bt cotton plant).
 4. Production of golden rice (rice having vitamin A) by incorporating
three genes required for its synthesis in rice plant.
 5. Bioremediation by the use of recombinant organisms and
 6. Use of genetic engineering techniques in forensic medicine.

22. Hormones
 Recombinant human insulin became the first
manufactured, or commercial, recombinant
pharmaceutical in 1982, when the FDA approved human
insulin for the treatment of cases of diabetes that require
the hormone.
 Before the development of recombinant human insulin,
animals (notably pigs and cattle) were the only
nonhuman sources of insulin. Animal insulin, however,
differs slightly but significantly from human insulin and
can elicit troublesome immune responses.
 The therapeutic effects of recombinant human insulin in
humans are identical to those of porcine insulin, and it
acts as quickly as porcine insulin, but its immune-system
side

23.  effects are relatively infrequent. Further, it can satisfy medical needs more
readily and more affordably. Other recombinant hormones include those
described below. Regular insulin ordinarily must be injected 30 to 45 minutes
before meals to control blood glucose levels. Lispro (Humalog)—a
recombinant insulinlike substance—is faster-acting than regular insulin.
Because injection of lispro is appropriate within 15 minutes before meals,
using it instead of regular insulin may be more convenient for some patients.
( ) Lispro.
 Erythropoietin (EPO), a hormone produced by the kidneys, stimulates the
bone marrow to produce red blood cells. The FDA has approved recombinant
EPO—epoetin alfa—for the treatment of anemia due to chronic renal failure.
Epoetin alfa.
 Human growth hormone (hGH) is used to counter growth failure in children
that is due to a lack of hGH production by the body. Before the introduction of
recombinant hGH the hormone was derived from human cadavers. Cadaver-
derived hGH was susceptible to contamination with slow viruses that attack
nerve tissue. Such infective agents caused fatal illnesses in some patients.
Recombinant hGH has greatly improved the long-term treatment of children
whose bodies do not produce enough hGH.

26. Recombinant human growth hormone.
 1. Clotting Factors
 Inadequate bodily synthesis of any of the many clotting factors can
prevent effective clotting. The FDA has approved two clotting-
related recombinant drugs: abciximab for the prevention of blood
clotting as
 an adjunct to angioplasty, and recombinant antihemophiliac factor
(rAHF) for the treatment of hemophilia A. Hemophilia A is a lifelong
hereditary disorder characterized by slow clotting and consequent
difficulty in controlling blood loss, even from minor injuries. About
20,000 persons in the United States alone have this condition,
which is due to a deficiency of antihemophiliac factor (AHF, or
 factor VIII). Before the introduction of rAHF, treatment of hemophilia
A required protein concentrates from human plasma. Such
concentrates could contain contaminants (e.g., HIV), and the
lifetime

27.  treatment of a single patient required thousands of blood
contributions. Persons with hemophilia B lack factor IX. They
require either factor IX concentrates from pooled human blood
or factor IX from cell cultures (some of which are genetically
engineered).

28.  Using Microbes Against Other Microbes
• Antibiotics
• Act in a few key ways
• Prevent replication
• Kill directly
• Damage cell wall or prevent its synthesis
Using Microbes for a Variety of Applications

29. Improving Antibiotic Production
 12,000 antibiotics have been identified
 Most from Gram-positive soil bacterium Streptomyces
 Some from fungi and other Gram-positive or Gram-
negative bacteria
 100,000 tons of antibiotics produced per year
 200-300 new discoveries per year
 Cost to bring new one to market very high
 1-2% prove useful
 Fleming’s fungal production 2 U/ml, now 70,000 U/ml
Biotechnology…

30. Cloning Antibiotic
Genes
 Mutate antibiotic
producing strain to
antibiotic negative
 Introduce plasmids
from genomic library
of wt strain
 Use clones to screen
large insert library
 Pathway may require
up to 20-30 steps…

31. Production of Penicillins
and Cephalosporins

32. Most Common Microbially Synthesized
Antibiotics

33. Antibiotics Produced by Strptomyces Strains and
Those Transformed by Plasmids Pij2303 and Pij2315

34. Vaccines
 In every modern vaccine the main or sole active ingredient consists of killed
microorganisms, nonvirulent microorganisms, microbial products (e.g., toxins), or
microbial components that have been purified. All these active ingredients are
antigens: substances that can stimulate the immune system to produce specific
antibodies. Such stimulation leaves the immune system prepared to destroy bacteria
and viruses whose antigens correspond to the antibodies it has learned to produce.
Although conventionally produced vaccines are generally harmless, some of them
may, rarely, contain infectious contaminants.
 Vaccines whose active ingredients are recombinant antigens do not carry this slight
risk. More than 350 million persons worldwide are infected with the virus that causes
hepatitis B, a major cause of chronic inflammation of the liver, cirrhosis of the liver,
and liver cancer. ( ) Hepatitis B kills a million people each year worldwide. About 1.25
million Americans harbor the hepatitis B virus (HBV);
 30 percent of them will eventually develop a serious liver disease. About 300,000
children and adults in the U.S. become infected with HBV each year, and 5,000
Americans die annually from liver disease

36. Some of the foreign genes that have been expressed in
recombinant vaccinia viruses.

37. Vaccines
 First was a vaccine against smallpox (cowpox
provides immunity)
• DPT-diphtheria, pertussis, and tetanus
• MMR –measles, mumps, and rubella
• OPV- oral polio vaccine (Sabin)

38.  A Primer on Antibodies
• Antigen- foreign substances that stimulate an immune
response
• Types of leukocytes or white blood cells
• B-lymphocytes: antibody-mediated immunity
• T-lymphocytes: cellular immunity
• Macrophages: “cell eating” (phagocytosis)
Vaccines

39. Heavy chain
Light chain
IgA – first line of defense
IgG and IgM – activates
macrophages
Vaccines
 Antigens stimulate antibody production in the immune system

40. Vaccines
 Mechanism of Antibody Action

41.  How are vaccines made?
• They can be part of a pathogen (e.g. a toxin) or
whole organism that is dead or alive but attenuated
(doesn’t cause disease)
• Subunit (toxin) or another part of the pathogen
• Attenuated (doesn’t cause disease)
• Inactivated (killed)
 What about flu vaccines (why do we have to get
a shot every year?)
Vaccines

42. Recombinant VaccinesRecombinant Vaccines
 Vaccines – provide immunity to infectious microorganisms
Attenuated Vaccine Inactivated Vaccine Subunit Vaccine

43. Recombinant VaccinesRecombinant Vaccines
 Recombinant Vaccines
• A vaccine produced from a cloned gene
Video: Constructing Vaccines

44. Recombinant VaccinesRecombinant Vaccines
 DNA vaccines
• Direct injection of
plasmid DNA
containing genes
encoding specific
antigenic proteins

45. Bacterial and Viral Targets for
Vaccines
 HIV

46. 46
Principles of Vaccination
 A vaccine renders the recipient resistant to
infection.
 During vaccination a vaccine is injected or given
orally.
 The host produces antibodies for a particular
pathogen.
 Upon further exposure the pathogen is
inactivated by the antibodies and disease state
prevented.
 Generally to produce a vaccine the pathogen is
grown in culture and inactivated or nonvirulent
forms are used for vaccination.

47. 47
Principles of Vaccination
 There are many disadvantages and they
include:
 Not all organisms can be cultured.
 The procedure is expensive and sometimes
unsafe.
 New pathogens keep occurring.
 For some pathogens e.g. HIV vaccination is
not appropriate.
 why?

48. 48
New Generation of Vaccines
 Recombinant DNA technology is being used to produce a
new generation of vaccines.
Virulence genes are deleted and organism is still
able to stimulate an immune response.
Live nonpathogenic strains can carry antigenic
determinants from pathogenic strains.
If the agent cannot be maintained in culture, genes
of proteins for antigenic determinants can be
cloned and expressed in an alternative host e.g. E.
coli.

49. 49
New Generation of Vaccines
 There are three types of vaccines we will be
discussing:
 Subunit (protein) vaccines
 Attenuated vaccines
 Vector vaccines
 Subunit Vaccines
 Antibodies usually bind to surface proteins of
the pathogen or proteins generated after the
disruption of the pathogen.
 Binding of antibodies to these proteins will
stimulate an immune response.
 Therefore proteins can be use to stimulate an
immune response.

50. 50
Principles of Vaccination
 It has been showed that the capsid or envelope
proteins are enough to illicit an immune response.
E.g:
 Herpes simplex virus envelop glycoprotein O.
 Foot and mouth disease virus capsid protein (VP1)
 Extracellular proteins produced by Mycobacterium
tuberculosis.

51. 51
A Subunit Vaccine for M. tuberculosis (e 2nd
)
 Tuberculosis is caused by Mycobacterium
tuberculosis.
 The bacterium form lesions in the tissues and
organs causing cell death. Often the lung is
affected.
 About 2 billion people are infected and there
are 3 million deaths/year.
 Currently tuberculosis is controlled by a vaccine
called BCG (Bacillus Calmette-Guerin) which is
a strain of M. bovis.
 M. bovis often responds to diagnostic test for M.
tuberculosis.

52. 52
A Subunit Vaccine for M. tuberculosis
 Six extracellular proteins of M. tuberculosis
were purified.
 Separately and in combinations these proteins
were used to immunized guinea pigs.
 These animals were then challenged with M.
tuberculosis.
 After 9-10 weeks examination showed that some
combinations of the purified proteins provided
the same level of protection as the BCG vaccine.

53. 53
Attenuated Vaccines
 Attenuated vaccines often consists of a
pathogenic strains in which the virulent genes are
deleted or modified.
 The Development of a Live Cholera Vaccine.
 Live vaccines are more effective than a killed or
subunit (protein) vaccines.
 With this in mind a live vaccine for cholera was
developed.
 Cholera is characterized by fever, dehydration
abdominal pain and diarrhea.

54. 54
A Live Cholera Vaccine
 The causal agent of cholera is Vibrio cholerae
and is transmitted through contaminated water.
 V. cholerae produces a enterotoxin with an A
subunit and 5 B subunits.
 Presently the cholera vaccine consist of a phenol-
killed V. cholerae and it only last 3-6 months.
 A live vaccine would be more effective.
 In the sequence of the A peptide a tetracycline
resistance gene is inserted.

55. 55
A Live Cholera Vaccine
 A plasmid with A peptide was digested with 2
restriction enzymes Cla1 and Xba1.
 This removes 550 bases of A peptide.
 A Xba1 linker was added and T4 ligase used to
ligate the DNA. This plasmid was mixed with V.
cholerae with tetracycline resistant gene.
 By conjugation the plasmid was transferred to
the strain with the tetR
gene inserted into it’s
chromosomal DNA.

56. 56
Production
of a Live
Cholera
Vaccine

57. 57
A Live Cholera Vaccine
 By recombination the A peptide with the tetR
gene was replaced by the deleted A peptide.
 The final result is V. cholerae with a 550 bp of
the A peptide deleted.
 If this can be used as a vaccine is being
tested.

58. 58
Production
of a Live
Cholera
Vaccine

59. 59
Vector Vaccine
 A vector vaccine is a vaccine which is introduced
by a vector e.g. vaccinia virus.
 The vaccinia virus as a live vaccine led to the
globally eradication of the smallpox virus.
 The genome of the vaccinia virus has been
completely sequenced.
 The virus replicates in the cytoplasm rather than
in the nucleus.
 The vaccinia virus is generally nonpathogenic.

60. 60
Vector Vaccine
 These characteristics makes the vaccinia virus a
good candidate for a virus vector to carry gene for
antigenic determinants form other pathogens.
 The procedure involves:
 The DNA sequence for the specific antigen is
inserted into a plasmid beside the vaccinia virus
promoter in the middle of a non-essential gene
e.g. thymidine kinase.

61. 61
Vector Vaccine
 The plasmid is used to transform thymdine
kinase negative cells which were previously
infected with the vaccinia virus.
 Recombination between the plasmid and
vaccinia virus chromosomal DNA results in
transfer of antigen gene from the recombinant
plasmid to the vaccinia virus.
 Thus virus can now be used as a vaccine for the
specific antigen.

62. 62
Insertion of antigen
gene into vaccinia
virus genome

63. 63
Vector Vaccine
 A number of antigen genes have been inserted
into the vaccinia virus genome e.g.
 Rabies virus G protein
 Hepatitis B surface antigen
 Influenza virus NP and HA proteins.
 A recombinant vaccinia virus vaccine for rabies is
able to elicit neutralizing antibodies in foxes
which is a major carrier of the disease.

64. Monoclonal Antibodies
 Other Biotech Drugs
 Listed below are a few of the hundreds of other biotech drugs that are either
in clinical use or undergoing scientific investigation.
 Biotech vaccines undergoing investigation include vaccines for acellular
pertussis (whooping cough), AIDS, herpes simplex, Lyme disease, and
melanoma.
 Two new recombinant interferons are undergoing investigation: consensus
interferon, for treating hepatitis C; and recombinant beta interferon 1a, for
multiple sclerosis.
 Recombinant PTK (protein tyrosine kinase) inhibitors may have therapeutic
utility against diseases marked by cell proliferation, such as cancer,
atherosclerosis, and psoriasis. Protein tyrosine kinases contribute to cell
division and are the targets of these biotech drugs.
 Recombinant human interleukin-3 is undergoing clinical investigation as an
adjunct to traditional cancer chemotherapy.
 Two recombinant growth factors (cytokines that regulate cell division) are
undergoing major clinical trials: recombinant human insulin-like growth
factor (rhIGF-1) and recombinant human platelet-derived
 growth factor-BB (PDGF). PDGF can contribute to wound healing.

65.  In December 1997 the FDA approved clinical testing of a recombinant version of the
cytokine myeloid progenitor inhibitory factor-1 (MPIF-1). MPIF-1 can keep certain
normal cells, including many immunologically important cells, from dividing and can
thus protect them from anticancer drugs that target rapidly multiplying cells. When
such anticancer drugs affect normal cells that divide rapidly, hair loss, nausea, and
immunosuppression can result.
 Injecting the recombinant protein fibroblast growth factor (FGF-1) into the human
myocardium increases the blood supply to the heart by inducing blood-vessel
formation. ( ) Such treatment, called a "biologic bypass" or "biobypass," does not
require surgery. FGF-1 is injectable nonsurgically into the myocardium by cardiac
catheterization. A biobypass may benefit persons with coronary artery disease whose
arteries are not reparable surgically. (A gene-therapy form of biobypass, VEGF gene
therapy, is described below.)
 In January 1998 advisors to the FDA recommended that the agency approve Apligraf,
a recombinant skin replacer, for the treatment of leg ulcers due to poor circulation;
and DermaGraft, another such product, for the treatment of diabetic ulcers. About
800,000 diabetic foot ulcers occur in the U.S. annually, and they lead to most of the
lower-leg amputations that approximately 60,000 diabetics

66. 66
Production of Monoclonal Antibodies
 Monoclonal antibodies results from a clone of a B
lymphocyte producing a single antibody which
will bind to a specific epitope of an antigen.
 What is a polyclonal antibody?
 Monoclonal antibodies are produced:
 Fusion of a myeloma (B cell which has become
cancerous) with a spleen cell that is immunized
with a specific antigen.
 The resulting hybridomas are tested for the
production of a monoclonal antibodies.

67. 67
Production of
Monoclonal
Antibodies

68. 68
Production of Human Monoclonal Antibodies by E. coli
 Hybridoma cells grow relatively slow and require
expensive media.
 To circumvent this problem human monoclonal
antibodies are grown in E. coli.
 The produce involves:
 mRNA is isolated from the B cell.
 cDNA is synthesized from the mRNA by the
enzyme reverse transciptase.
 Both heavy and light chains are amplified
separately from the cDNA using PCR.
 The amplified products are cut with restriction
enzymes and cloned into Lambda vector.

69. 69
Production of
Human
Monoclonal
Antibodies by
E. coli

70. 70
Production of Human Monoclonal Antibodies
by E. coli
 During cloning different light and heavy chains
are cloned.
 The DNA of one heavy and one light chain are
cloned into the same vector.
 Many different combinations of H and L chains
are cloned together in the same vector.
 Lambda is not useful for producing large
amounts of proteins.
 The L and H chains are excised from Lambda
and cloned into an E. coli plasmid and the
recombinant plasmid transformed into E. coli.

71. 71
Production of
Human
Monoclonal
Antibodies
by E. coli

72. 72
Production of Human Monoclonal Antibodies
by E. coli
 E. coli will produce large amounts of
monoclonal antibodies which are
harvested.
 These monoclonal antibodies can be used
for:
 Diagnostic purposes e.g detection of HIV by
ELISA.
 Therapeutically for the treatment of infection.

73. Biosynthesis of Amino Acids
 Amino acids uses in food industry
 Flavor enhances
 Antioxidants
 Nutritional supplements
 Amino acid uses in agriculture
 Feed additives
 Amino acid uses in medicine
 Infusion solutions
 Amino Acid uses in industry
 Starting materials for polymer and cosmetic
production

74. Commercial
Applications of
Amino Acids

75. Cysteine Biosynthesis by E. coli
 Serine acetyltransferase is
feedback inhibited
 Site-directed mutagenesis
 Transform into E. coli
strain that does not
degrade cysteine
 Even better feedback
insensitive enzyme genes
isolated as cDNAs from
Arabidopsis and
transformed into strain

76. 76
Enzymes as Therapeutic Agents/ DNase1
• Cystic fibrosis (CF) is one of the most fatal heredity
diseases among European and their descendants with
~30,000 cases in the US and 23,000 in Canada.
• Furthermore among European descendants it occurs in 1
in 2,500 live birth and 1 in 25 are carriers.
• It is caused by more than 500 different mutations in the
cystic fibrosis transmembrane conductance regulator
(CFTR) gene.
• Individuals with CF are highly susceptible to bacterial
infection and antibiotic treatment often results in
resistant strains.

77. Thanks
for your
attention

78. A practical example: Manufacturing human insulin
• Insulin a hormone that regulates the level of
sugar in the blood
• • People with defective genes for insulin
have diabetes and must take insulin shots
• • Before recombinant DNA, insulin was
obtained from animal tissue:

79. • Goal:
• to insert human insulin gene into a
bacteria so that the bacteria can produce it
for our use.
• Relatively inexpensive (already done)

80. Steps for making insulin
• Plasmids are obtained from bacteria cells, cleaved with a
restriction endonuclease
• • Human chromosomes (DNA) are collected and cleaved
with the same restriction enzyme
• – many fragments of chromosomal DNA are formed, but
only some of the fragments contain the insulin gene.
• • Chromosomal and plasmid DNA fragments are mixed
together with enzyme called DNA ligase

81. • recombinant plasmids are then put back
into bacteria, yeast or some other rapidly
dividing type of cell
• • We screen our library to identify
which bacterial colonies contain
recombinant plasmids with the insulin
gene --

82. • We now have a collection of recombinant
DNA plasmids
• – some contain the insulin gene, while
others do not
• – this collection of plasmids is called a
DNA library.

83. Therapeutic proteins
• Recombinant insulin in bacteria
Using Microbes for a Variety of Applications

84. 84
Nucleic Acids as Therapeutic agents (e 3rd
)
• Many human disorders e.g. cancer and inflammatory
conditions (virus, parasites) are often caused by
overproduction of a normal protein.
• Theoretically a small ss nucleic acid can hybridize to a
specific gene or mRNA and diminish transcription or
translation.
• An oligonucleotide (oligo) that binds to a gene and
blocks transcription is an antigene.
• An oligo that binds to mRNA and blocks translation is
called an antisense oligo.
• Ribozyme (catalytic RNA) and interfering RNA
( RNAi) can target specific mRNA for degradation.

85. 85
Antisense RNA

86. 86
Antisense Oligonucleotide

87. 87
Antisense RNA
• Episomally based expression vectors with cDNA for
insulin-like growth factor 1 (ILGF-1) receptors were
constructed in the antisense version.
• ILGF-1 is prevalent in malignant glioma a common
form of brain cancer and prostate carcinoma.
• Culture of glioma cells when transfected with the
antisense version of ILGF-1 in ZnSO4lost its tumurous
properties.
• A similar treatment of mice which were injected with
prostate carcinoma cells caused small or no tumor to
develop.

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Antisense Oligonucleotides
• Antisense deoxynucleotides can also be used
as therapeutic agents.
• However when injected into the body is
deoxynucleotides are susceptible to
degradation.
• To prevent this modified deoxynucleotides are
used including phosphorothioate,
phosphoramidate and polyamide.
• Free oligos are usually introduced into to the
body encapsulated in a liposome.

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Modified Deoxynucleotides
Phosphodiester linkage Phosphorothioate linkage

90. 90
Modified Deoxynucleotides
Phosphoramidite linkage Polyamide linkage

91. 91
Liposome

92. 92
Therapeutic Nucleic Acids
• Several preclinical trials have been conducted
with antisense oligos.
• Narrowing of the coronary and carotid arties can
lead to heart attacks and strokes.
• This condition is alleviated by angioplasty.
• This involved inserting a balloon into the
blocked artery and inflating it.
• This often results in injury of the artery and
subsequent healing can result in blockage in 40%
of patients within 6 months.

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Therapeutic Nucleic Acids
• When antisense oligo that target the mRNA for a
protein essential for the cell cycle was applied to rat
carotid arteries after angioplasty the reoccurring
blockage was reduced by 90%.
• Furthermore smooth muscle cell proliferation is
implicated in other disease such as:
Ω Atherosclerosis
Ω Hypertension
Ω Diabetics mellitus
Ω Failing of coronary bypass graft
• Similar antisense therapeutics could be used to help
alleviate these conditions.

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Antisense Oligos and Psoriasis
• Antisense oligos have also been tested in the treatment
of psoriasis.
• Psoriasis is uncontrollable epidermal growth.
• ILGF-1 receptors are implicated in the pathogenesis of
psoriasis.
• 15 nt antisense oligo were transferred into keratinocytes
using liposome and the amount of ILGF-1 protein was
decreased by 45-65%.
• When mouse with human psoriasis lesions were
injected with anitsense oligi complementary to ILGF-1
receptor mRNA there was significant reduction (58-
69%) in epidermal thickness.

95. 95
Interfering RNA
• The addition of dsRNA to an animal cell causes the
degradation of the mRNA from which it is derived.
• This process is called gene silencing or RNA inference
(RNAi).
• Gene silencing has been shown to be a natural
mechanism which plant and animals use to protect
against viruses.
• The dsRNA that is introduced is cleaved by dicer-like
dsRNAse into ssRNA of 21-23 nt.
• These short oligos complex with RISC ( RNA inference
inducing silencing complex) which degrade the mRNA
complimentary to the oligos.
• This process can be used to target specific mRNA.

96. 96
RNA1 as Therapeutic Agents
• A viral vector was used to deliver a small
fragment of RNA to brain cells of mice with
SCA1 (human neurodegenerative disease
spinocerebellar ataxia 1).
• This suppress the SCA1 gene and the mice has
normal coordination and movement.
• Scientists are optimistic about using RNAi to
treat other neurological diseases such as
Alzheimer’s and Hunting’s disease.

97. 97
HIV Therapeutic Agents (e 2nd
)
• Acquired immune deficiency syndrome (AIDS) is
caused by the human immunodeficiency virus
(HIV).
• The target of HIV are the T helper cells (TH).
• TH cells play a pivotal role in the immune system
by the release of cytokines which stimulate other
immune cells.
• The gp120 glycoprotein of HIV binds to CD4
receptors of TH cells.
• The THcells become infected with the virus and
are destroyed, slowly shutting down the immune
system.

98. 98
Interaction of HIV with CD4

99. 99
HIV Therapeutic Agents
• HIV antiviral strategies may include:
• Production of antibodies to CD4 (will block CD4
receptors on TH cells and prevent infection by HIV).
• Production excess CD4 protein (react with gp120
protein therefore HIV cannot infect TH cells).
• Both strategies do not destroy HIV but only block
infection.
• To stop HIV infection we need to develop strategies
which will destroy HIV.

100. 100
HIV Therapeutic Agents
• One strategy which will protect TH cells and destroy
HIV include the production of a fusion protein.
• The fusion protein will have 2 parts CD4 protein
attached to the Fc portion of an immunoglobulin
(CD4 immunoadhesion).
• The CD4 portion will attach to the gp120 protein of
HIV or virus infected cells.
• The immunoglobulin portion will initiate a cytotoxic
response to destroy the virus or virus infected cell.

101. 101
CD4
Immunoadehesion
Fusion Protein

102. 102
HIV Therapeutic Agents
• Another strategy involves making a second fusion protein.
• The CD4 sequence is ligated to the sequence of Pseudomonas
exotoxin A to form a fusion protein.
• HIV infected cells have gp120 proteins on their surfaces.
• The CD4 portion of the fusion protein will attach to the infected
cells.
• The fusion protein will enter the cells and initiate the killing of
the infected cell.
• Pseudomonas exotoxin A inactivates the protein synthesis by
affecting elongation factor EF-2. This prevents further protein
synthesis and eventually causes death of the infected cell.

103. 103
CD4-Pseudomonas Exotoxin Fusion
Protein