This document discusses the production of insulin and human growth hormone (HGH) using recombinant DNA technology. It describes how conventional methods of extracting these proteins from animal sources are expensive and can cause allergic reactions. Recombinant DNA methods involve isolating the genes for insulin and HGH and inserting them into organisms like E. coli to produce the proteins. For insulin, different methods include producing the A and B chains separately and joining them, or inserting the proinsulin sequence. Analog insulin production also modifies the amino acid sequence. HGH is important for growth and development, and its gene has been inserted into expression cassettes and cultivated in bioreactors. Pharming is also discussed as a method of producing proteins by

1. Insulin and HGH production
using rDNA Technology
Mrinal Vashisth

2. Conventional Methods of recombinant
protein production v/s rDNA Methods
• Conventional methods fail to meet the supply
and demand
• Conventional methods are much more expensive
on a larger scale as compared to rDNA methods
• Conventional methods include plant/animal
harvesting of the compound – this may cause
allergic reactions or even diseases
• rDNA methods involve isolating the gene of
interest and inserting it in the organism to get the
desired protein usually with or without the help
of a vector

3. Insulin: Importance
• Insulin is a hormone produced in
pancreas by the islets of Langerhans.
• Regulates the amount of glucose in the
blood
• Malfunctioning of insulin production
leads to a condition called diabetes
• Diabetes are of two types:
(a.) Type I: When no insulin is produced
at all
(b.) Type II: When insulin produced is in
less quantity than what is required
• There are two chains A and B that
constitute Human Insulin
• Chain A: 21 amino acids; Chain B: 30
amino acids
• Both chains could be separately
produced as preproinsulin – modified
into proinsulin by combining A and B
• Proinsulin when attached to a signal
molecule becomes the functional insulin

4. Conventional methods of Insulin
Production
• Frederick G. Banting and Charles H. Best first
purified insulin in 1921 from Dog’s pancreas
• Later methods were developed for isolation of
Insulin from cattle and pigs’ pancreas –
administration however resulted in allergic
reactions in some.
• 1982– Eli Lily produced Insulin using E. coli strain
– Insulin thus became the first genetically
engineered pharmaceutical product.

5. rDNA Technology used for Insulin
Production
-Method I: A and B chains are produced separately in
different in separate bioreactors
o They are joined using lysosomes and cyanogen
bromide
o Purification by chromatography
-Method II: Proinsulin process: The sequence coding for
proinsulin is inserted in a non-pathogenic strain of E.
coli.
o After fermentation, insulin is isolated and additives like
Zn2+ are added which delay the absorption in body
-Method III: Analog insulin (Glargin insulin) production:
Amino acid sequence is changed
o This analog clumps less and disperses more readily in
the blood.

7. HGH: Importance
• Secreted by pituitary gland,
responsible for normal body
growth and development
• Development of muscles, bones,
lengthening of height etc. are all
regulated by HGH
• Low production of HGH results in
Dwarfism while High production of
HGH results in giant stature
• Children of age 1 were treated with
GH from deceased humans
• This therapy continues for eight
years but there is a risk of
transmission of diseases

8. • It has a 191 amino
acids long single chain
• There are disulphide
bonds in 2 peptide
loops
• 2 HGH genes are there
in humans:
(1.) hGH-N
(2.) hGH-V

9. Method of Production
• hGH cDNAs are constructed
• Expression cassettes with hGH cDNAs insert is
constructed
• Recombinant clones are cultivated in small
scare flasks/ bioreactors
• Producing the hGH in pilot scale bioreactors
• Developing large scale purification procedure
and process chromatography optimization
(Affinity chromatography)

11. Pharming as a method of producing
recombinant proteins
• Using genetic engineering making host animals
produce a pharmaceutical compound (recombinant
protein or their metabolic product) they wouldn’t
otherwise produce is called pharming.
• The resulting animal is thus a Genetically Modified
Organism (GMO)
• Advantage over bioreactors is that Pharming doesn’t
require expensive infrastructre – can easily be scaled
up as per demand and supply
• Also, complex proteins which microbial systems simply cannot
produce can be produced by means of pharming
• There are however a lot of ethical issue raised by
activists that impede this method
• E.g. Genzyme - antithrombin III in goat milk

12. Method of Pharming

13. THANK YOU!

14. References
• Pharming for Farmaceuticals,
http://learn.genetics.utah.edu/content/scienc
e/pharming/, Retrieved on April, 15, 2017
• Demain L. A, Vaishnav P, Production of
Recombinant Proteins by Microbes and Higher
Organisms, Biotechnology Advances 27 (2009)
297–306, 10.1016/j.biotechadv.2009.01.008