insulin production using rdna technology-.pptx

insulin production using rdna technology

1. Insulin and HGH production using rDNA Technology

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 They are joined using lysosomes and cyanogen bromide Purification by chromatography - Method II: Proinsulin process: The sequence coding for proinsulin is inserted in a non-pathogenic strain of E. coli. 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 This analog clumps less and disperses more readily in the blood.

6. 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

7. 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

8. 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)

9. 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

10. Method of Pharming

11. THANK YOU!

12. References Pharming for Farmaceuticals , http://learn.genetics.utah.edu/content/science/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