APPLICATIONS OF GENETIC ENGINEERING

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GURMEET SINGH M.PHARMACY CTIPS

GENETIC ENGINEERING
The manipulations of genes at genomic level in-vitro is known as Genetic Engineering. The use of experimental techniques to produce DNA molecules containing new genes or new combinations of genes. Also called Recombinant DNA Technology or Genetic Modification, is the direct human manipulation of an organism's genetic material in a way that does not occur under natural conditions.

The most common form of genetic engineering involves the insertion of new genetic material at an unspecified location in the host genome. This is accomplished by Isolating and copying the genetic material of interest

Generating a construct containing all the genetic elements for correct expression

Inserting this construct into the host organism.

PRODUCTION OF PROTEINS OF PHARMACEUTICAL SIGNIFICANCE

These include Insulin Human Growth Hormone Tissue Plasminogen Activator Hepatitis-B Vaccine Interferons

INSULIN

Human Insulin was the first recombinant-derived product, used for the treatment of individuals suffering from diabetes. Insulin is a protein, which is naturally obtained from -cells of the Islets of Langerhans of the Pancreas. It is a molecule with 51 amino acids in two polypeptide chains (A-chain = 21 and B-chain = 30 ), held together by disulphide cross linking.

INSULIN PRODUCTION
Chemical synthesis of genes i.e. chain A and B Both the chains cloned separately and attached to a plasmid near the end of -galactosidase end of E.coli Two bacterial strains were constructed, each producing a fused protein with A and B chains The genes chemically synthesized were devoid of promoters, ribosomal binding sites and initiation codon (Methionine residue)

(To express the cloned genes into functional protein, it is essential to clone a gene into plasmid vector closed to bacterial promoter and having ribosomal binding site and initition codon )

The authentic insulin can be obtained by cleaving the methionine residue by treatment with cyanogen bromide

Purification and the two chain were linked chemically in vitro to produce insulin

HUMAN GROWTH HORMONE

Growth hormone (GH) is a protein-based peptide hormone. It stimulates growth, cell reproduction and regeneration in humans and other animals. Growth hormone is a 191-amino acid, single-chain polypeptide that is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. Somatotropin refers to the growth hormone 1 produced naturally in animals, whereas the term Somatropin refers to growth hormone produced by recombinant DNA technology and is abbreviated "HGH" in humans

SOMATOSTATIN

Being a very short protein, only 14 amino acids in length. It was ideally suited for artificial gene synthesis. The strategy used was the same as described for recombinant insulin, involves insertion of the artificial gene into a lac Z vector, synthesis of fusion protein, and cleavage with cyanogen bromide. Finally purification and storage of hormone.

SOMATOTROPHIN

Somatotrophin is 191 amino acid long, which is used to treat GH deficient children. A combination of artificial gene synthesis and complementary DNA cloning was used to obtain a somatotrophin-producing E.coli strain. E.coli involves the assembly of a gene that is partly synthetic and partly natural.

Complementary DNA prepared from HGH mRNA is found to have a single cleavage site in the sequence coding for amino acids 23 and 24 of HGH. Following the cleavage with restriction enzymes, the large DNA fragment (amino acid 24-191) is combined with a chemically synthesized DNA fragments (amino acid 1-23) containing an ATG (initiation codon). The complete gene is inserted into a modified version of pBR322 incorporating the lac operon.

Fermentation in E.coli host is followed by isolation of the product from intact cell. (if additional methioninyl residue attached to the HGH, these should be removed because they cause immunogenecity)

THANK YOU
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