Biotechnology techniques are applied in the production of pharmaceutically important products. These pharmaceutical products can be used for the improvement of quality of life of humans. Most important application of biotechnology technique called as recombinant DNA technology is the production of most important biomolecules such as insulin, follicle stimulating hormones, blood products like clotting factor VIII, albumins, tissue plasminogen activators and also cytokines and also interferons, colony stimulating factors and much more.
Tissue Plasminogen Activator:
Tissue plasminogen activator is a protease enzyme, helps in dissolving blood clots. In normal condition Plasmin degrades fibrin and hence dissolves blood clots. The Plasmin is produced by activation of plasminogen by tissue plasminogen activator.
Tissue plasminogen activator has got very high therapeutic value, as this can be used in removing arterial thrombi or in other words blood clots. Hence protects heart and brain from blood clots.
Production of Recombinant Tissue Plasminogen Activator:
1. Recombinant DNA technology is used in producing cDNA molecule complementary to the mature mRNA of gene encoding tissue plasminogen activator.
2. This cDNA is then cloned into a synthetic plasmid and introduced into mammalian cells.
3. Transformed mammalian cells are cultured in a suitable animal cell culture media
4. Tissue plasminogen activator producing cells are then transferred into fermenters to produce them in large quantity.
5. Tissue plasminogen activator secreted into the culture media is isolated and purified.
6. Tissue plasminogen activator is the first pharmaceutical product produced using mammalian cell culture. Example is ACTIVASE, second generation tissue plasminogen activator are called as AITEPLASE and RETEPLASE
Uses of Tissue Plasminogen Activator:
1. Tissue plasminogen activator dissolves blood clots and degrades it without reducing the blood clotting capability in other parts of the body.
2. Tissue plasminogen activator can be administered intravenously
3. Action of tissue plasminogen activator is faster than any other thrombolytic agents.
4. Tissue plasminogen activator shows very less side effects.
Interferons are produced by a cell infected by a virus. Interferons are proteins which help in protecting other healthy cells from viral infection. In other words, proteinaceous interferons are secreted by human cell to resist the immediate invasion of virus to neighbouring healthy cells and also to resist abnormal cell multiplication.
Interferons are used as therapeutic agent against many viral diseases like common cold and hepatitis. Biotechnology technique or recombinant DNA technology is used to produce recombinant interferons with enhanced specific activity (antiviral activity).
Production of Recombinant Interferon:
1. cDNA is produced complementary to the mature mRNA encoding for a specific interferon.
2. This cDNA is integrated with plasmid
3. This recombinant plasmid is then introduced into E.coli cell
4. These transformed bacterial cells are cultured in large industrial fermenters using suitable culture media
5. Interferons are isolated from the culture media and are purified using appropriate method.
Mammalian cells like mouse fibroblast cell culture or human leukocyte culture are used for the production of interferons in large quantity. As production of interferons is relatively less in bacterial cells, because most of the interferon molecules are glycoprotein in nature and bacterial cells does not have the machinery for glycosylation or post translational modification.
Interferon production using Yeast Cells:
Yeast cells are the perfect host for the production of functional interferons as they possess the mechanism to carry out post translational modifications such as glycosylation of protein molecules.
1. DNA sequence encoding for human interferon is isolated
2. This coding DNA sequence is cloned to yeast alcohol dehydrogenase gene in a plasmid vector.
3. This plasmid is then introduced into yeast cells.
4. These recombinant cells are culture in large industrial fermenters and produced interferons are isolated and then purified.
5. In the same way hybrid interferons that are interferons with different antiviral activities are combined to produce hybrid interferons.
Production of Interferon-β:
1. Human fibroblasts are used in the production of Interferon-β biomolecules.
2. Human Interferon-β gene is isolated and incorporated into a plasmid vector.
3. This plasmid vector is then incorporated into E.coli cells.
4. Transformed E.coli cells are selected and cultured in large industrial fermenters.
5. Produced Interferon-β is extracted and purified using appropriate purifying method.
1. Interferons are used in the treatment of large number of viral diseases.
2. Interferons are used in the treatment of cancer.
3. Interferons are used in the treatment of common cold and also influenza
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