Insulin production and synthesis
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The document discusses insulin, including its structure, gene location, processing in the body, and treatment of diseases related to insulin deficiency. Insulin is produced in the pancreas and enables cells to take up glucose. The human insulin gene is located on chromosome 11. Insulin is composed of two peptide chains linked by disulfide bonds. Treatment for insulin deficiency involves injection of insulin extracted from animals or recombinant human insulin produced using biotechnology. Recombinant human insulin is made through bacterial or yeast expression systems using vectors like plasmids or yeast artificial chromosomes.
Insulin production and synthesis
- 1. INSULIN By: Nancy Saber Roba shaat Mohamed El-Asaly Under the supervision of: Prof. dr. Aziza mahrous Prof. dr. Ahmed abd elmaksoud
- 3. • Introduction • Structure of the insulin • Insulin gene • Insulin processing in the body • Disease • Treatment • Recombinant Insulin and process • Host cells • Vectors • Bioreactors
- 4. 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.
- 7. Which Chromosome Insulin Gene Is Found On? • INS: Insulin gene is found on chromosome (11). • It is found on the short arm of chromosome 11 ( p arm). • It is located region 1 , band 5, sub- band 5.
- 8. The Structure Of Insulin • Insulin is composed of two peptide chains referred to as the α chain and β chain. • α and βchains are linked together by two disulfide bonds, and an additional disulfide is formed within the α chain. • α chain consists of 21 amino acids and the β chain of 30 amino acids.
- 11. Failure To Produce Or Use Insulin
- 12. Importance Of Insulin • Without insulin, the blood glucose builds up in the blood and the cells are starved of their energy source. • Some of the symptoms that may occur include fatigue, constant infections, blurred eye sight, numbness, tingling in the hands or legs, increased thirst, and slowed healing of bruises or cuts. • The cells will begin to use fat, the energy source stored for emergencies. • When this happens for too long a time the body produces ketones, chemicals produced by the liver. • Ketones can poison and kill cells if they build up in the body over an extended period of time. This can lead to serious illness and coma.
- 15. TREATMENT • Banting and Best invented the insulin hormone in 1921. • Several individuals died since their glucose level increased due to disorder of insulin production . • Treatment via insulin injection extracted from animal pancreas.
- 16. PROBLEM? Animal Insulin swine and cow. Synthetic Human Insulin Slight difference in composition As a consequence • Human body produces antibodies against the animal insulin; inflammation at the area of injection. • Long-term complications occurred • Decrease in animal insulin production
- 17. New technology Introduced through the new field of Biotechnology Recombinant DNA technology
- 18. THE BREAKTHROUGH •Recombinant human insulin :a form of insulin (trade name Humulin) made from recombinant DNA which is human insulin.
- 20. RECOMBINANT INSULIN APPROACHES Eli Lilly ( bacterial host) Novo nordisk (Yeast host)
- 21. HOST CELL USED FOR INSULIN PRODUCTION
- 22. ADVANTAGES OF USING E.COLI AS A SYSTEM • Simple, well-understood genetics • Ease of genetic manipulation • Minimal culturing cost • Fast expression (doubling time is only 20 - 30 mins). • Well established labeling protocols for stability studies. • Established regulatory track record. • Fermentation: ease of scaling up. • Ease of Inclusion bodies purification.
- 23. DISADVANTAGES OF USING E.COLI AS A SYSTEM • Loss of plasmid and antibiotic property • Unsolicited inducers for gene expression • Intracellular accumulation of heterologous proteins as inclusion bodies • Improper protein refolding • Lack of post- translational modifications (including unable to form disulphide bonds) • Protein-mediated metabolic burden and stress • Endotoxin contamination • Poor secretion • Proteolytic digestion complexity in downstream processing
- 24. EXPRESSION VECTOR • A circle of double- stranded DNA that is separate from the chromosomes, which is found in bacteria.
- 25. ADVANTAGES OF USING BACTERIAL PLASMID •Small, easy to handle •Straightforward selection strategies •Useful for cloning small DNA fragments •(< 10kbp)
- 26. DISADVANTAGES OF USING BACTERIAL PLASMID •Can’t uptake large DNA molecules.
- 28. HOST CELL USED FOR INSULIN PRODUCTION •Saccharomyces cerevisiae is an eukaryotic microbe system that is widely used for protein expression that require post-translational modification.
- 29. ADVANTAGES OF USING SACCHAROMYCES CEREVISIAE • Nonpathogenic • Rapid growth (generation time ca. 80 min) • Dispersed cells • Ease of replica plating and mutant isolation • Can be grown on defined media giving the investigator complete control over environmental parameters • Well-defined genetic system • Highly versatile DNA transformation system
- 30. DISADVANTAGES OF USING SACCHAROMYCES CEREVISIAE •S. cerevisiae is not as productive as the E. coli.
- 31. EXPRESSION VECTOR USED IN SACCHAROMYCES CEREVISIAE • Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone DNA sequences in bacterial and yeast cells. • It is a plasmid vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. • Circular form when they are amplified or manipulated in E. coli • Rendered linear and of very large size when introduced as cloning vectors in yeast
- 32. YAC CONSTITUENTS It consists of several important regions • TEL: the telomere which is located at each chromosome end, protects the linear DNA from degradation by the nucleases. • CEN: the centromere which is the attachment site for the mitotic spindle fibers , “pulls” one copy of each duplicated chromosome into each new daughter cell. • ORI: the replication origin sequences which are specific DNA sequences that allow DNA replication machinery to assemble on the DNA . • - It is also know as ARS: autonomously replicating sequence vectors in yeast
- 33. YAC CONSTITUENTS It also consists of specific sequences: • Yeast selectable marker A and B: Selectable markers allow easy isolation of yeast cells that have taken up the artificial chromosome. • Bacterial selectable marker: Such as ampicillin resistance ; for screening and selection purposes. • Recognition site: For restriction enzyme ligation such as EcoR1 , BamHi
- 34. ADVANTAGES OF YAC • Extremely large DNA molecules (up to more than 1 Mb) can be introduced and propagated in the form of yeast artificial chromosomes (YACs) in S. cerevisiae. • It can be used to express eukaryotic proteins that require posttranslational modification. • Screening and selection properties. • YACs can be utilized to clone and assemble the entire genomes of an organism. • Physical mapping.
- 35. DISADVANTAGES OF YAC • Low transformation efficiencies. • YACs are very difficult to manipulate. • Approximately 40 % of the YACs from most libraries are deleted. • Approximately 40-60% of the YACs from most libraries are chimeric.
- 36. Scaling up
- 37. PHASES INVOLVED IN INSULIN PRODUCTION Upstream Process Downstream Process
- 38. UPSTREAM PROCESS PHASE Shaking bioreactor Advantages: Easy ,Visible ,Cheap, Depyrogenation feasible Disadvantage: Poor aeration Impeller jams Requires cleaning siliconizing & sterilization lHigh space requirements in incubator
- 39. UPSTREAM PROCESS PHASE Stir tank bioreactor • Continuous bioreactor processes continually feed nutrients and medium into the bioreactor while also continually harvesting material from the bioreactor.
- 40. UPSTREAM PROCESS PHASE Stir tank bioreactor • Agitator is introduced to disperse the reactants thoroughly into the reaction mixture immediately as they enter the reactor. • Product is continuously drawn out and that’s why known for perfect mixing. • Compositions at outlet and inside reactor are the same.
- 41. ADVANTAGES OF STIR TANK BIOREACTOR • Multi-gas and pH control • Increased Capacity( 5 L to 500 L) • Versatility
- 42. DISADVANTAGES OF STIR TANK BIOREACTOR • Costly • High power due the presence of mechanical pumps • Limitation of Weight Preparation • Requires siliconizing • Requires constant cleaning, • Sterilization, depyrogenation • Requires high Maintenance -Chiller, parts
- 43. BIOREACTOR CONDITIONS •10L total working volume •31 hour growth phase •pH 7 •37 °C
- 44. DOWNSTREAM PROCESS • Removal of insolubles is the first step and involves the capture of the product as a solute in a particulate-free liquid. Typical operations to achieve this are filtration, centrifugation. • Product isolation is the removal of those components whose properties vary considerably from that of the desired product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.
- 45. DOWNSTREAM PROCESS • Product purification is done to separate those contaminants that resemble the product very closely in physical and chemical properties. operations include affinity, size exclusion, reversed phase chromatography, ion-exchange chromatography, crystallization and fractional precipitation. • Product polishing describes the final processing steps which end with packaging of the product in a form that is stable, easily transportable and convenient.