RECOMBINANT INSULIN AND GROWTH HORMONE PRODUCTION

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IT HAS VERY DETAIL ABOUT INSULIN AND GROWTH HORMONE PRODUCTION

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RECOMBINANT PRODUCTION OF INSULIN AND GROWTH HORMONE PRESENTED BY: GOPINATH.V.E. DEPARTMENT OF PHARMACOLOGY SHREE DEVI COLLEGE OF PHARMACY KENJAR MANGALORE

CONTENTS: 

CONTENTS INSULIN INTRODUCTION STEPS IN PRODUCTION GROWTH HORMONE INTRODUCTION STEPS IN PRODUCTION

Diabetes: 

Diabetes INTRODUCTION Types of diabetes:- Type I- Here no insulin is produced Type II- Here insulin is produced in little quantity than actually needed

INSULIN- INTRODUCTION: 

INSULIN- INTRODUCTION Insulin is a hormone that regulates the amount of glucose in the blood Insulin is produced by cells in the pancreas, called the islets of Langerhans . Insulin is of three types :- Ultra short acting Short acting Intermediate acting Long acting

Miles stones passed in insulin production: 

Miles stones passed in insulin production In 1921, Canadian scientists Frederick G. Banting and Charles H. Best successfully purified insulin from a dog's pancreas. Later Insulin was extracted from the pancreas of cattle and pigs and purified which produce immune reactions in some patients In 1982, the Eli Lilly Corporation produced a human insulin that became the first approved genetically engineered pharmaceutical product.

Materials used for insulin: 

Materials used for insulin Human insulin is grown in the lab inside common bacteria. Escherichia coli Human protein that produces insulin containing about 20 amino acids Large fermentation tanks to grow the bacteria Nutrients needed for the bacteria Centrifuge, various chromatography and x-ray crystallography instruments.

Structure of Insulin: 

Structure of Insulin The insulin gene is a protein consisting of two separate chains of amino acids, an A above a B chain, that are held together with bonds. The insulin A chain consists of 21 amino acids and the B chain has 30. Before becoming an active insulin protein, insulin is first produced as preproinsulin . After preproinsulin , the chain evolves into proinsulin , still a single chain but without the signaling sequence. Active protein insulin, the protein without the section linking the A and B chains.

Types of Insulin Production: 

Types of Insulin Production Type-I Here two insulin chains are grown separately and inserted into plasmids and grown in E.coli . Then 2 chains are joined by oxidation-reduction reaction using lysosomes and cyanogen bromide Then purified by chromatography and other techniques.

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Type-II it is also known as Proinsulin process The sequence that codes for proinsulin is inserted into the non-pathogenic E. coli bacteria and fermentation is carried out. Additives like Zn2+ delay absorption in the body Type-III Analog insulin is produced by changing its amino acid sequence and creating an analog. Which clumps less and disperses more readily into the blood. Ex:- Glargine insulin

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Strategy for insulin production

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Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

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Human Insulin Production by Bacteria 6) join the plasmid and human fragment and cut with a restriction enzyme

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Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences The enzyme EcoRI cutting DNA at its recognition sequence Different restriction enzymes have different recognition sequences. This makes it possible to create a wide variety of different gene fragments. Restriction enzyme animation

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Human Insulin Production by Bacteria Mix the recombinant plasmid with bacteria. Screening bacterial cells to learn which contain the human insulin gene is the hard part.

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Route to the Production by Bacteria of Human Insulin A fermentor used to grow recombinant bacteria. This is the step when gene cloning takes place. The single recombinant plasmid replicates within a cell. Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene. One cell with the recombinant plasmid

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Route to the Production by Bacteria of Human Insulin The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.

Insulin crystals from the purification process : 

Insulin crystals from the purification process

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NEW DEVELOPMENT- INSULIN PENS Owen Mumford Ltd., UK

Future Insulins: 

Future Insulins Jet injectors Controlled Insulin pumps Oral insulin pills Insulin inhaler Insulin patches

Human growth hormone (hGH, or Somatotropin) : 

Human growth hormone (hGH, or Somatotropin) Secreted by the pituitary gland, and is responsible for normal body growth and development. These processes together are responsible for longitudinal growth,production in muscle and bones ,break down of fats etc Inadequate production of GH results in short stature, defined as a below normal height for a given age

Traditional treatment of Dwarfism: 

Traditional treatment of Dwarfism Children were treated with injection of GH from deceased humans. No other animals GH were used in contrast to Insulin. Atleast 8 cadavers were needed to treat a child of age 1 which is continued for 8-10 years There is also chance of transmission of diseases

Growth Hormone: 191 amino acids, single chain: 

Growth Hormone: 191 amino acids, single chain Structure of human Growth hormone Teritiary structure Primary structure Hormone binding to receptors

Structure of growth hormone: 

Structure of growth hormone It composed of 191 amino acid residue linked by disulfide bridges in 2 peptide loops 2 hGH genes are seen in human 1. hGH -N 2.hGH-V

Production of recombinant GH: 

Production of recombinant GH Isolating and constructing hGH cDNAs Constructing expression cassette with hGH cDNAs inserts Cultivating the recombinant clones in small scale flask/bioreactor Producing the hGH in pilot scale bioreactors Developing large scale purification procedure and process chromatography optimization (Affinity chromatography )

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Strategy for production of growth hormone

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DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways These are recombinant DNAs and they often are made of DNAs from different organisms.