description of recombinant proteins insulin and growth hormone by recombinant DNA technology

1. PRODUCTION OF HUMAN INSULIN USING RECOMBINANT
MICRO ORGANISMS
D.INDRAJA

2. Introduction
• Synthesis of cellular proteins is ultimately under the control of genes, any defect in the gene produces no
protein (or) incorrect protein(some times it may be ineffective or it may cause disease)
• This leads to genetically linked diseases
• RDNA technology can be fruitfully employed to produce
human proteins that can be used for the treatment of genetically linked diseases
• The earliest uses of biotechnology in pharmaceutical manufacturing is the use of recombinant DNA
technology to modify Escherichia coli bacteria to produce human insulin, which was performed at
Genentech in 1978
• Insulin is a hormone produced by β-cells of islets of Langerhans of pancreas.
• It was discovered by sir Edward Sharpey Schafer (1916) while studying Islets of Langerhans
• People who do not produce the necessary amount of insulin have diabetes.

3. INSULIN
• Insulin Is a polypeptide hormone produced by the β cells of the islets of Langerhans in the pancreas
• Its main function is enabling the cells to take up glucose ( providing it with energy it needs).
• It is required for normal glucose homeostasis.

4. •Chemically Human insulin is small, simple protein compo
sed of 51 amino acids sequences and has a molecular weight
of 5808 Da.
•Insulin hormone is a dimer of a A- chain and a B-chain
which are linked together by a disulphide bond.
Fredrick Sanger et al (1954) gave the first complete
description of insulin. Insulin consists of two polypeptide
chain,
• Chain A- 21 amino acids long
• Chain B-30 amino acids long
Both chains are joined together by disulphide bond
between two cysteine residue
Structure of Human Insulin:

5. Diabetes mellitus
• It is a genetically linked disease characterized by increased
blood glucose conc(hyper glycemia)
• The occurrence of diabetes is due to inefficient or insufficient
insulin production
• As insulin facilitates the cellular uptake and utilization of
glucose for release of energy .due to inefficient one it can not
take glucose in to the cell from blood stream this leads to
increased conc of glucose exceeds about 180 mg/dl then it is
excreted through urine
• The patients with diabetes are weak and tired since the
production of energy ATP is much depressed
• This leads to nephropathy(kidney), neuropathy(nerves),
retinopathy(eyes), and circulatory diseases
• This is the third leading cause of heart attack where heart
diseases and cancer are first and second
• They need insulin therapy to keep healthy
Diabetes mellitus

7. Conventional method
• In early years insulin isolated and purified from the pigs and cows and used for the patients
• There is a slight difference in their structure when compared to human insulin
Drawback
• It results in allergies in some of the diabetic patients
• Moreover large number of animals are sacrificed to obtain the product, almost 70 pigs must be
sacrificed to obtain insulin for the treatment of diabetic patient for just one year

8. Production of insulin in general
• In pancreatic cells insulin is produced from pre proinsulin mRNA as a pre proinsulin polypeptide
• The pre sequence containing leader or signal sequence is cleaved off as the protein secreted outside the
cell membrane
• The resulting proinsulin is folded and linked by three interchain disulphide bonds
• Then the middle portion is cleaved by enzyme peptidases then it results in production of active insulin

9. Production of recombinant insulin
• Attempts for the production of insulin started in 1970’s
• In 1980’s recombinant insulin was injected to treat diabetes at guy’s hospital London and it was
succeeded
• In 1982 approval by concerned authorities was given to produce recombinant insulin
• In 1986 Eli lilly company produced human insulin under the trade name of humulin

10. Production of recombinant insulin from microorganisms (method-1)
• In the early eighties, human insulin produced by recombinant technology entered the pharmaceutical
market.
• In one of the approaches the sequences for the A and B chains were synthesized chemically and inserted
separately downstream of the β -galactosidase structural gene controlled by the X lac promoter
• The construction was such that the insulin chains would be made as fusion proteins joined by a
methionine to the end of the β -galactosidase protein and induced with IPTG(mimic of allolactose)
• The expression vector also contained an Amp’ marker. The transformants were then screened by plating
on a culture medium containing X-gal and ampicillin.
• The insulin A chain and B chain transformants were grown to harvest the cells in large quantity.
• The cells were lysed and the insulin A chain and B chain were purified separately.
chains
chains

11. • A gene was fused to the β-galactosidase gene, therefore the insulin protein produced was therefore a β -
galactosidase-insulin hybrid
• It is treated with Cynogen bromide which acts as a peptidase and finally the chains are isolated and
purified and chemically combined to produce active insulin

12. (Method-2)
• A more effective method, also using a strain of E. coli, involves the production of proinsulin completely
whole, instead of separate fragments of insulin synthesis.
• Proinsulin consists of chains A and B, connected with chain C .
• The gene encoding proinsulin cloned to E. coli cells is also expressed there.
• The resulting hormone is cleared and then the chain C is proteolytically removed

13. (Method-3)
• Human insulin mRNA was isolated from pancreatic cells and copied into proinsulin cDNA and it is
tagged with both start codon(ATG) and stop codon(TGA) and introduced in to bacterial plasmid with
fusion peptide
• And the cell is induced with IPTG and after the expression it is treated with cyanogen bromide and
proinsulin is formed and with the help of peptidase active insulin was obtained

14. Second generation recombinant insulin
• After injecting insulin plasma concentration of insulin rises slowly, so it has to be injected 15 min before
a meal
• Further decrease in the injected insulin level is also low leads to production of hyperinsulinemia this is
due to therapeutic insulin exist as hexamer which dissociates slowly to biologically active dimer or
monomer
• Attempts has done to produce second generation insulin by site directed mutagenesis and protein
engineering these are called muteins  these fastly dissociates in to active form
• Example  insulin Lispro with modified amino acid residues at position 29 and 30 of B-chain of insulin
and it can also be injected immediately before a meal
• Insulin lispro, sold under the brand name Humalog among others, is a type of insulin used to treat type 1
and type 2 diabetes. Typically it is taken around the time of eating

15. Chemically altered porcin insulin
• Porcin insulin differs from human insulin just by one amino acid alanine in place of threonine at C
terminal of B-chain
• Attempts have made to alter the chemical structure of porcin insulin and this modified one is used for
treatment of diabetes mellitus

16. PRODUCTION OF HUMAN GROWTH
HORMONE USING RECOMBINANT
MICRO ORGANISMS

17. Introduction
• Growth hormone (GH) or somatotropin, also known
as human growth hormones (hGH or HGH) in its human
form, is a peptide hormone that stimulates growth, cell
reproduction, and cell regeneration in humans and other
animals.
• It is thus important in human development. GH also
stimulates production of IGF-1 (Insulin-like growth factor
1 (IGF-1), also called somatomedin C, is a hormone similar
in molecular structure to insulin which plays an important
role in childhood growth, and has anabolic effects in
adult)and increases the concentration of glucose and free
fatty acids.
• Growth hormone is produced by the pituitary gland. It
regulates the growth and development. Growth hormone
stimulates overall body growth by increasing the cellular
uptake of amino acids , and protein synthesis, and promoting
the use of fat as body fuel

18. • Insufficient human growth hormone (HGH) in young children
results in retarded growth, clinically referred to as pituitary
dwarfism.
• while High production of HGH results in giant stature
• Growth hormone is used as a prescription drug in medicine to
treat children's growth disorders and adult growth hormone
deficiency.
• In the United States, it is only available legally from
pharmacies, by prescription from a doctor.
• In recent years in the United States, some doctors have started
to prescribe growth hormone in GH-deficient older patients (but
not on healthy people) to increase vitality.
• While legal, the efficacy and safety of this use for HGH has not
been tested in a clinical trial

19. Abnormally tall stature.
Abnormal growth of the face, hands and feet Irregular menstrual cycle
Thickened facial
features
Deafness
Double vision
Delayed puberty
Excessive perispiration with
slight activity
Symptoms of
Gigantism

20. Symptoms of
Dwarfism
Very short trunk
Short neckShortened arms and legs
Broad, rounded chest.
Opening in the roof of the mouth (cleft palate)
Hip deformities that result in thighbones turning inward
Slightly flattened
cheekbones

21. • In addition to increasing height in children and adolescents, growth hormone has many other effects on
the body:
* Increases calcium retention, and strengthens and increases the mineralization of bone
* Increases muscle mass through sarcomere hypertrophy
* Promotes lipolysis
* Increases protein synthesis
* Stimulates the growth of all internal organs excluding the brain
* Plays a role in homeostasis
* Reduces liver uptake of glucose
* Promotes gluconeogenesis in the liver
* Contributes to the maintenance and function of pancreatic islets
* Stimulates the immune system
Structure
• human growth hormone is a protein of 191 amino acids
and a molecular weight of 22,124 daltons.
• The structure includes four helices necessary for functional
interaction with the GH receptor.

22. Conventional method
• Before the invention of rDNA technology, HGH could only be produced laboriously by isolating it from
pituitary gland tissue taken from human cadavers.
• This process was inefficient, expensive and sometimes unsafe.
• For example, the resulting HGH product occasionally contained contaminants from cadaver tissues.
• Rarely, patients injected with HGH from cadavers developed Creutzfeld-Jakob disease, a very serious
human version of mad cow disease.
• Infection is caused by proteins called prions.
• By eliminating the need for human tissue, rDNA technology avoids these and other potential
contamination problems.

23. Production of growth hormone in general
• During the course of its natural synthesis in the body., HGH is tagged with a single peptide (with 26
amino acids) The signal peptide is removed during secretion to release the active HGH for biological
functions.
• The entire process of HGH synthesis goes on in an orderly fashion in the body.
191 A.A
Signal peptide(26 A.A)
Active human growth hormone

24. Production of recombinant HGH from microorganisms
• Biotechnologists can now produce HGH by genetic engineering.
• The technique adopted is quite comparable with that of insulin production.
• The procedure essentially consists of inserting HGH gene into E.coli plasmid, culturing the cells and
isolation of the HGH from the extracellular medium
• Somatrem was first marketed under the brand name Protropin by Genentech in 1985.
• Humatrope is a man-made form of human growth hormone. It was first approved in 1987 to treat
children who are growing slowly because they do not make enough growth hormone on their own by
Elililly
Limitation in HGH production
• signal peptide interrupts HGH production by recombinant technology.
• The complementary DNA (cDNA) synthesized from the mRNA encoding HGH is inserted into the plasmid.
• The plasmid containing E.coli when cultured, produces full length HGH along with signal peptide.
• But E.coli cannot remove the signal peptide. Further, it is also quite difficult to get rid of signal peptide by various
other means.
• Theoretically, cDNA encoding signal peptide can be cut to solve these problems.
• Unfortunately, there is no restriction endonuclease to do this job, hence this is not possible.

25. A novel approach for
HGH production:
• Biotechnologists have resolved the problem of
signal peptide interruption by a novel approach.
• The base sequence in cDNA encoding signal
peptide ( 26 amino acids ) plus the neighbouring 24
amino acids is cut by restriction endonuclease
ECoR I.
• Now a gene (cDNA ) for 24 amino acid sequence of
HGH is freshly synthesized and ligated to the
remaining HGH cDNA.
• The so constituted cDNA , attached to a vector, is
inserted into a bacterium such as E. coli for culture
and production of HGH.
• In this manner, the biologically functional
HGH can be produced by recombinant DNA
technology.

26. Use of recombinant growth hormone in animals
• It is now available for administration to farm animals to promote early growth and development
• Such farm animals yield linear meat, besides increased milk production
• However the use in farm animals is a controversial issue
Use of recombinant human growth hormone (hGH)
1.Treatment of children suffering from growth deficiency
2.Treat the patient with Turner’s syndrome and chronic renal insufficiency
3.To treat patient with renal carcinoma
4.Bovine somatotropin hormone is used to increases milk production in lactating cows and also to increase
body mass of cattle's