genetic engineer applications

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APPLICATIONS OF GENETICENGINEERING

GURMEET SINGHM.PHARMACY

CTIPS


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GENETIC ENGINEERING

The manipulations of genes at genomic level in-vitro is known asGenetic Engineering.

The use of experimental techniques to produce DNA moleculescontaining new genes or new combinations of genes.

Also called Recombinant DNA Technology or Genetic

Modification, is the direct human manipulation of an organism'sgenetic material in a way that does not occur under naturalconditions.


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The most common form of genetic engineering involves theinsertion of new genetic material at an unspecified location in thehost genome.

This is accomplished by

Isolating and copying the genetic material of interest

Generating a construct containing all the genetic elements forcorrect expression

Inserting this construct into the host organism.


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PRODUCTION OF PROTEINS OFPHARMACEUTICAL SIGNIFICANCE

These include

Insulin Human Growth Hormone Tissue Plasminogen Activator Hepatitis-B Vaccine

Interferons


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INSULIN

Human Insulin was the first recombinant-derived product, usedfor the treatment of individuals suffering from diabetes.

Insulin is a protein, which is naturally obtained from -cells ofthe 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 disulphidecross linking.


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INSULIN PRODUCTION

Chemical synthesis of genes i.e. chain A and B

Both the chains cloned separately and attached to a plasmid nearthe end of -galactosidase end of E.coli

Two bacterial strains were constructed, each producing a fusedprotein with A and B chains

The genes chemically synthesized were devoid of promoters,ribosomal binding sites and initiation codon

(Methionine residue)


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(To express the cloned genes into functional protein, it is essentialto 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 methionineresidue by treatment with cyanogen bromide

Purification and the two chain were linked chemically in vitro toproduce insulin


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http://en.wikipedia.org/wiki/Peptide_hormone


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HUMAN GROWTH HORMONE

Growth hormone (GH) is a protein-based peptide hormone. Itstimulates growth, cell reproduction and regeneration in humansand other animals.

Growth hormone is a 191-amino acid, single-chain polypeptidethat is synthesized, stored, and secreted by the somatotroph cellswithin 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 growthhormone produced by recombinant DNA technology and isabbreviated "HGH" in humans
http://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormone


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SOMATOSTATIN

Being a very short protein, only 14 amino acids in length.

It was ideally suited forartificial gene synthesis.

The strategy used was the same as described for recombinantinsulin, involves insertion of the artificial gene into a lac Zvector, synthesis of fusion protein, and cleavage with cyanogen

bromide.

Finally purification and storage of hormone.


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SOMATOTROPHIN

Somatotrophin is 191 amino acid long, which is used to treatGH deficient children.

A combination of artificial gene synthesis and complementaryDNA cloning was used to obtain a somatotrophin-producingE.coli strain.

E.coli involves the assembly of a gene that is partly syntheticand partly natural.


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Complementary DNA prepared from HGH mRNA is found tohave a single cleavage site in the sequence coding for aminoacids 23 and 24 of HGH.

Following the cleavage with restriction enzymes, the large DNAfragment (amino acid 24-191) is combined with a chemicallysynthesized DNA fragments (amino acid 1-23) containing anATG (initiation codon).

The complete gene is inserted into a modified version of pBR-322 incorporating the lac operon.

Fermentation in E.coli host is followed by isolation of theproduct from intact cell.

(if additional methioninyl residue attached to the HGH, theseshould be removed because they cause immunogenecity)


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