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Universit De JIJEL Facult des Sciences Dpartement de Biologie Molculaire et Cellulaire

Recombinant DNA Technology in the Synthesis of Human Insulin

Presented by Boudjelida Houria Bellal Chafika Kaaboub Kawter 2010

Since Banting and Best discovered the hormone, insulin in 1921.diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.

The structure of insulin

Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains (see fig. 1) are linked by a disulfide bond.

The Vector (Gram negative E. coli)

A weakened strain of the common bacterium, Escherrichia coli (E. coli) , an inhabitant of the human digestive tract, is the 'factory' used in the genetic engineering of insulin.

Steps of insulin synthesis

I. Isolation of proinsulin gene The genes responsible for the synthesis of insulin are extracted from pancreatic cells. In the first step the genetic information for the synthesis of insulin transcribed to mRNA. By using an enzyme called reverse transcriptase, that mRNA is supplied with necessary nucleotides to produce a copy of complementary polynucleotide strand which resembles the DNA strand which codes for insulin.

The single stranded DNA is converted into a double-stranded helix. The double stranded DNA produced is called complementary DNA (cDNA). The cDNA thus formed is called proinsulin gene. It is with only exons. The proinsulin gene is linked to lac operon with B-galactosidase which provides initiation and termination signals for synthesis of insulin.

2.Insertion of proinsulin gene into plasmid The plasmid called pBR322 of E. coli used as a vector or molecular carrier. The restriction endonuclease called Hind III used to cleave the plasmid. The proinsulin gene is mixed with cleaved plasmid in the presence of enzyme DNA ligase. The restriction enzyme breaks the tetracycline resistant gene of plasmid. The proinsulin gene is inserted into this region. Now the plasmid with inserted proinsulin gene is called recombinant DNA.

3.Transfer of recombinant plasmid into host cell E. coli bacteria are generally used as host cells in insulin synthesis. They are treated with cold calcium chloride and immediately temperature increased to 42C. It results in destabilization of cell membrane and development of pores in that. Such cells are mixed with recombinant plasmid. This results in the migration of recombinant plasmids into the host cell. It results in bacterial transformation.

4. Cloning of proinsulin gene Before cloning of gene, the E. coli cells with recombinant DNA must be known. They can be easily identified as they are sensitive to tetracycline because in recombinant plasmid tetracycline gene is damaged and contains proinsulin gene. Such E. coli cells are selected and grown in agar medium to get large number of similar cells with recombinant plasmid. It results in cloning of proinsulin gene which are then used in insulin production.

5. Insulin production E. coli cells with proinsulin gene in their plasmids are grown in bioreactors containing nutrient medium. Addition of lactose into the bioreactor increases the production proinsulin. It acts as inducer, activates lac operon linked to proinsulin gene. The product is treated with cyanogen bromide (CNBr) to remove B-galactosidase protein. Proinsulin naturally folds, so the cysteine residues are opposite each other and the chemical treatment then causes the formation of disulfide cross-links.

Once this has been accomplished the connecting midchain c can be removed by treatment with proteolytic enzymes trypsin and carboxypeptidase B, which have no effect on insulin itself.

It results in the formation of active insulin.

Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely identical to that of the natural molecule. Initially the major difficulty encountered was the contamination of the final product by the host cells, increasing the risk of contamination in the fermentation broth. When the final insulin product is subjected to a battery of tests.