Applications of recombinant technology

1. APPLICATIONS OF R-DNA
TECHNOLOGY
BY
DILIP O. MORANI
ASST. PROF.
SHRI D. D. VISPUTE COLLEGE OF PHARMACY
AND RESEARCH CENTER, PANVEL
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2. Introduction
Development of recombinant technology created enormous potential for the
pharmaceutical industries.
• Cloned genes commercially utilized in pharmaceuticals for the production of
valuable products.
• Genes are responsible for the expression of proteins and these proteins and
peptides can be easily prepared by using recombinant technology.
• Recombinant proteins provide high level of sensitivity and specificity compared
with natural proteins which are isolated from plants, animals and micro-
organisms.
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• Biological activity of expressed protein depends on recombinant culture,
type, choice and characters of host cell.
• The production of recombinant proteins by transgenic animals is most
important application of r-DNA technology and can also be used for the
preparation of pharmaceutical proteins.
• Important biologicals prepared by r-DNA technology includes human
insulin, hepatitis B vaccine, interferons, tissue plasminogen activator,
erythropoietin, human growth hormone, interleukins etc.
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4. Important r-proteins & their applications
Sr. No. Proteins/Peptides Applications
1 Human insulin Diabetes mellitus
2 Hepatitis B vaccine Vaccination
3 Interferons Hepatitis, Cancer
4 Tissue plasminogen activator Anticoagulant
5 Erythropoietin Anaemia
6 Human growth hormone Pituitary dwarfism
7 Interleukins-1,2,3 Immune disorder & tumours
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5. Human Insulin
• Smallest protein secreted by Islet of Langerhans of pancreas which
catabolizes glucose in blood.
• Used for patients suffering from diabetes mellitus who are unable to
metabolize sugar.
• First derived from E. coli using r-DNA technology in 1982 for human use.
• Basic principle consist of inserting human insulin gene and promoter gene
of lac operon on the plasmids of E. coli.
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• It is composed of two polypeptide chains (A & B) linked via disulphide bonds.
• Polypeptide chain A contains 21 amino acids and chain B contains 30 amino acids.
• Insulin chains (A & B) are derived from preproinsulin which is synthesized in β-cells
of Islets of Langerhans containing 109 amino acids.
• When preproinsulin passes through cell membrane of synthesizing cells, they
delinked first 23 amino acids and forms proinsulin containing 86 amino acids.
• Finally, proinsulin converted to insulin by proteolytic enzymes such as
endopeptidase and thiol activated carboxypeptidase.
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8. Preparation of insulin
• It is prepared by r-DNA technology from synthetic gene or from mRNA
separated from rat pancreas.
• In 1977, Itakura et al chemically synthesized DNA sequence for A & B
chains of insulin.
• Synthetic A & B genes are separately inserted into 2 pBR plasmids by the
side of β -galactosidase gene.
• R-plasmids are separately transferred into E. coli cells.
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• Bacterial cells are grown in large fermenter by optimizing physical conditions
and using proper nutrients.
• The product contains large chimeric protein consisting of A chain or B chain
attached to naturally occurring E. coli protein.
• These 2 chains (A & B) can be obtained by detaching from β-galactosidase
through cyanogen bromide.
• Chain A and chain B are combined to form insulin by sulphonating the two
peptides with sodium sulphite and sodium disulphonate.
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• In recent years, second generation insulin are produced by protein
engineering and site directed mutagenesis.
• Second generation r-proteins are termed as muteins.
• Large number of insulin muteins have been prepared with the objective of
faster dissociation of hexamers to biologically active forms.
• Insulin lispro is prepared with modified amino acid residues at position 29
and 30 of B-chain of insulin.
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• Porcin (Pig) insulin is different from human insulin by just one amino acid
i.e. alanine in place of threonine at the C-terminal end of B-chain of human
insulin.
• Researcher have developed methods to alter the chemical structure of
porcine insulin to make it similar to human insulin.
• Chemically modified porcine insulin can also be used for the treatment of
diabetes mellitus.
• It is estimated that clones of E. coli are capable of producing about one
million molecules of insulin per bacterial cell.
• Human insulin is the first therapeutic product produced by recombinant
technology by Eli Lilly and company.
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13. Interferons
• Antiviral substance and first line of defense against viral attacks.
• In 1957, Alec Issacs and Jean Lindermann, the two British scientists
discovered a glycoprotein produced by cells called interferon.
• Set of small proteins which are secreted by cell in response to viral
infections.
• Human interferons are about 145 amino acid long and its molecular weight
range in between 20,000-30,000 Daltons.
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14. Types of interferons
• Interferons are classified into 3 types based on their physicochemical and
antigenic properties.
1. Alpha interferon (IFN-α) or leucocyte interferon
2. Beta interferon (IFN-β) or fibroblast interferon
3. Gamma interferon (IFN-γ) or immune interferon
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15. Mechanism of action
• Interferons inhibit viral replication and protect the cell from other
intracellular parasites.
• It activates natural killer cells and macrophages, stimulates B cells and
increases cell resistance to many microbial infections.
• Also, they have a significant role in the treatment of hepatitis B, cancer and
other viral diseases.
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16. Production of interferon
• Human interferon genes are inserted in E. coli for production of interferon
by r-DNA technology.
• cDNA was synthesized from mRNA of a specific interferon.
• This is then inserted into a plasmid vector and transferred to the cells.
• The interferon can be isolated from culture medium.
• Interferons are glycoprotein in nature and thus, their production in bacterial
cell is relatively less.
• Bacterial cell do not possess the machinery for glycosylation of proteins.
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• Yeast is most suitable for production of recombinant interferons.
• Yeast cells possess the mechanism to carry out glycosylation of proteins.
• DNA sequence coding for human leucocyte interferon is attached to the
yeast alcohol dehydrogenase gene in a plasmid.
• R-plasmid is introduced into yeast cells of Saccharomyces cerevisiae.
• In yeast cell, plasmid grows easily to replicate into glycoproteins.
• Plasmids are successfully replicate in E. coli but production is slow as
compared to yeast cells.
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• Hybrid interferons are more reactive in performing their functions and can
be produced by creation of hybrid genes from the genes of different
interferons.
• These genes are digested by restriction endonucleases and resulting
fragments are ligated to generate hybrid genes.
• Appropriate hybrid genes can be selected and transferred to bacterial cell to
produce hybrid interferons.
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19. Hepatitis B vaccine
• Hepatitis B virus is one of at least three hepatitis viruses causing a systemic infection
with pathological changes in the liver (hepatitis A, hepatitis B, and non-A, non-B
hepatitis).
• Hepatitis B is the most important of all the viral hepatitis.
• It accounts for half of all clinical hepatitis seen in some countries and is responsible
for much of the mortality, with an acute case fatality rate of about 1%.
• From 5% to 100 of patients infected with hepatitis B become chronic carriers.
• In addition to the disability associated with the acute clinical disease, chronic liver
disease, cirrhosis and hepatocellular carcinoma are now recognized sequelae of
unresolved hepatitis B infection.
• Indeed, in some areas of Asia and sub-Saharan Africa, hepatocellular carcinoma,
ostensibly attributable to hepatitis B infection, ranks as a leading cause of cancer
deaths among males. 19

20. Introduction
• Infection is transmitted to susceptible persons through close contact with the blood,
or other body fluids of chronic infectious carriers or persons suffering acute infection.
• The incubation period for hepatitis B is long.
• Six weeks to six months may elapse between exposure to infection and onset of
clinical symptoms.
• The illness usually begins with fatigue and anorexia and sometimes is accompanied by
myalgia and abdominal discomfort.
• Later, jaundice, dark urine, light-coloured stools, and tender hepatomegaly may appear.
• In other cases, the onset may be rapid, with appearance of jaundice early, in
association with fever, chills, and leukocytosis. 20

21. Hepatitis B virus
• Hepatitis B virus (HBV) has been identified as a 42-nm particle containing
double-stranded DNA.
• Infection with HBV is manifested by at least three antigenic markers:
hepatitis B surface antigen (HBsAg), the core antigen (HBcAg), and the e
antigen (HBeAg), resulting from replication of the virus in the hepatocytes.
• The surface antigen is found as 18-22-nm spherical particles (sometimes
slightly larger or smaller) and as tubular forms, and possesses a common
determinant a and generally, at least two mutually exclusive subdeterminants
d or y and w or r
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• Hepatitis B virus (HBV) is one of the most common infectious diseases
known to man.
• The World Health Organization (WHO) estimates that there are as many
as 285 million chronic carriers of this virus worldwide.
• Hepatitis B is 50 to 100 times more infectious than AIDS.
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24. Hepatitis B R-Vaccine
• It’s a novel and significant developed vaccine which is produced from the
antigenic proteins of Hepatitis B virus by recombinant process that
duplicates the chemical messages and secreted factors (Interleukin-2) for
the communication and activity of immune cells.
• A special type of tropical monocot banana under the genus Musa in the
Musaceae family, can be ideally engineered by genetic mechanism process
for the production of hepatitis B vaccine--- it was suggested.
• With this technology, the cost of vaccination could be reduced.
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• Suraface antigen
HBsAG is found as
18-22 nm spherical
or tubular form
particles. Recently
HBsAg gene or it’s
subunits are used
for the production
of recombinant
Hepatitis B
vaccine. 25

26. Criteria for production of Hepatitis B vaccine
• After infection in human being, HBV fails to multiply and infect a large number of
cells and even does not grow in cultured cells.
• Rather, HB viral antigen is obtained from the plasma of infected persons.
• This property has been explained to be due to hindrance of its molecular
characterization and expression, and thus helps in the development of vaccines.
• Although the whole viral genome can be cloned and sequenced, yet there is limited
information about amino acid sequence of surface and core antigens.
• Recently, HBV DNA has been successfully cloned in E. coli and mammalian cells,
and synthesis of HBsAg and HBcAg particles has been done in the cells.
• In 1981, HBcAg genes were inserted in PBR322 near β- galactosidase gene.
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27. Benefits of R-Hepatitis B vaccine
• Traditional vaccines use a weakened or killed form of a virus to force the
body to develop antibodies that are strong enough to combat the virus.
• Using recombinant DNA technology, the vaccine uses the surface antigen
of the virus that stimulates the production of protective antibodies which
combat the HB virus.
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28. Steps for production of Hepatitis B vaccine
• Production of these genes is needed in order to get production of vaccines on a
large scale.
• A general procedure for the production of recombinant Hepatitis B vaccines are
described here-
1. HBs antigen producing gene is isolated from the HB virus by normal isolation
process (cell lysis, protein denaturation, precipitation, centrifugation and drying).
2. A plasmid DNA is extracted from a bacterium- E.coli and is cut with restriction
enzyme- Eco RI forming the plasmid vector.
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3. The isolated HBs antigen producing gene is located and inserted into
the bacterial plasmid vector on forming the recombinant DNA.
4. This recombinant DNA, containing the target gene, is introduced into a
yeast cell forming the recombinant yeast cell.
5. The recombinant yeast cell multiplies in the fermentation tank and
produces the HBs antigens.
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6. After 48 hours, yeast cells are ruptured to free HBsAg.
The mixture is processed for extraction.
7. The HBs antigens are purified.
8. HBsAg are combined with preserving agent and other ingredients and
bottled.
Then, it is ready for vaccination in humans. 30

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32. REFERENCES
• Pharmaceutical biotechnology- Fundamentals and Applications by Prof.
Chandrakant Kokare, Sixth edition-August 2018, Nirali Prakashan, page no.
13.1 to 13.5.
• Hepatitis B vaccines prepared from yeast by recombinant DNA techniques:
Memorandum from a WHO Meeting.
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33. THANK YOU
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