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Production of Recombinant Human Growth Hormone SomatotropinBY: Nidhi Uppangala | Category: Biotechnology-products | Submitted: 2010-08-27 10:43:10
Recombinant DNA technology is widely used in the production of therapeutic agents such as hormones, cytokines, growth factors, antibiotics, vaccines, blood products like albumin, thrombolytics, fibrynolytics, clotting factors such as factor VII, factor IX, tissue plasminogen activator and many more. All these therapeutic agents can be produced in a large quantity and also more economically by using recombinant DNA technology.
Growth hormones are produced in the pitutary gland, and are involved in the overall growth of the body by increasing the production of protein, thus increase the amino acid uptake by the body cells. Growth hormone also increases the use of fat as body fuel.
Recombinant human growth hormone Somatotropin Production:
Recombinant human growth hormone is generally produced by inserting the human growth hormone gene into plasmids of E.Coli bacteria. Recombinant bacterial cells are cultured and human growth hormones produced by these bacteria are extracted from the extracellular media.
Human growth hormones are also produced and extracted using animal cell culture are now a days are used in treating patients with renal carcinoma and also to treat children who are suffering from human growth deficiency
During the production of the human growth hormone in the body it is attached with signal peptide with 26 amino acids. This signal peptide is cleaved in the body before releasing the human growth hormone to do their biological function.
During the production of recombinant human growth hormone the signal peptides interrupts their production. First cDNA is produced using mRNA encoding for human growth hormone. Then cDNA are integrated into the plasmid and are inserted into the E. Coli bacteria, then they are cultured and human growth hormone are extracted along with the signal peptide chain. As E.Coli bacteria does not have the mechanism to cleave signal peptide chain.
1. To resolve the interruption of signal peptides in the production of human growth hormone, the base sequence coding for signal peptides of around 26 amino acids along with neighbouring 24 amino acids is cut in the coding cDNA using restriction enzyme EcoR1.
2. Deleted 24 amino acid sequence of human growth hormone cDNA is freshly prepared and ligated to the remaining human growth hormone cDNA.
3. This coding cDNA is then integrated into the plasmid, then inserted into E.Coli bacterial cell
4. These recombinant bacterial cells are cultured, and then human growth hormones are extracted from the media.
Production of Recombinant Human Growth Hormone in Chinese Hamster Ovary (CHO) Cells:
This approach has got some advantages such as recombinant human growth hormone is secreted directly into the protein free media. Hence hormone can be extracted and purified using simple methods, and this also avoids resolubilization of inclusion bodies and refolding of proteins.
Extraction of Somatotropin:
Somatotropin is a growth hormone contains 191 amino acids and produced by the anterior pitutary or in other words front section of the pitutary gland. This hormone is activated by another hormone produced in the liver known as somatomedin. Thereby somatotropin causes growth.
2. In the case of human growth hormone somatotropin, the protein is sequestered in refractile bodies within the cytoplasm of the body cells.
3. Refractile bodies can be recovered from the cell culture by disrupting the cell to release the refractile bodies. These refractile bodies are then collected as pellet by using differential centrifugation method.
4. These refractile bodies are solubilized using suitable chotropic agents, such as urea or guanidine hydrochloride at pH 9-12 or alkaline pH.
5. Solubilized proteins are then treated with oxidizing agent, and this forms intramolecular disulfide bondsand protein is refolded to form its biologically active structure.
6. Biologically active somatotropins are used to enhance the growth of animals, and also animal productivity by administering these hormones by subcutaneous injection or intramuscular injection. They can also be admistered as implants.
Somatotropin is also used to treat patients with Turner's syndrome and also in chronic renal insufficiency.
8. Bovine somatotropin is used effectively to increase milk production in lactating cow.
9. Porcine somatotropin is used effectively to improve feed efficiency and to increase meat content, by administering it to finishing hogs.
Some of the examples of recombinant human growth hormone are
Protropin produced by Genetech Company
Humatrope produced or manufactured by Eri Lilly Company
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