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Overview:

Insulinimage

Structure of Insulin, made of 51 amino acids

Insulin is a a small and simple protein consisting of 51 amino acids, 30 of which comprise one polypeptide chain, and the remaining 21 comprising a second chain; the two chains are linked by a disulfide bond. A hormone protein produced by Beta-cells in the pancreas, inulin's main purpose is in the regulation of blood sugar levels. Insulin is released after a meal, and is the body's way of communicating to the cells and skeletal muscle that glucose is now present in the bloodstream, and must be taken up for use. Insulin also makes sure that there is not too much excess glucose circulating through the blood, and is thus crucial for maintaining homeostasis, as prolonged hyperglycemia can cause blindness and other neurological damage.

Medicinal Use of Insulin:

Insulinactions

People who are diabetic either do not produce enough insulin on their own (Type 1 diabetes) or are insulin-resistant (Type II diabetes). Both forms of diabetes require the administration of external (synthetic) insulin, usually in the form of injection. Whereas in the past, human diabetics were injected with cow and pig insulin, this form of therapy often proved dangerous, as many patients had severe adverse reactions to the non-human insulin. Today, with modern medicine and genetic techniques, recombinant DNA technology has allowed for insulin to be made in the lab and periodically injected into humans subcutaneously or via an insulin pump (continuous subcutaneous flow of insulin).

Process of Making Synthetic DNA via Bacteria: Recombinant DNA Construct, Cells Used, Purification:

The most common vector used to host and synthesize human insulin is the bacteria E. coli, and the process is as follows:

1. The gene for producing human insulin is isolated. Since the gene is part of the normal human DNA found in a human chromosome, the gene can be easily-isolated and copied so that multiple rounds of synthesis can occur.

2. Insulin has two chains--A and B (see image above)--and DNA polymerase is used to make a complimentary strand of A and a complimentary strand of B.

Plasmid

3. These now-double-stranded DNA fragments are inserted into plasmids

4. Each DNA fragment is inserted into the Beta-galactosidase gene on the plasmid, which effectively acts as the human beta cells in the pancreas would, serving as the site for expression of the insulin protein. Each plasmid is also equipped with a tetracycline-resistant gene (explanation to follow)

5. The plasmids are transformed into bacteria(E.coli), and tetracycline is added to kill any untransformed bacteria. The bacteria undergo the following synthesis of insulin while submerged in a broth containing nutrients for optimal growth.

6. The transformed bacteria are grown and, over time, the Beta-galactosidase and insulin fusion protein secreted by the beta cells are harvested and purified.

7. Purification of the protein sees that the Beta-galactosidase portion is cleaved and discarded, so one is left with just the insulin protein.The initial step in the purification scheme, however, is to separate the bacteria from the broth in which it undergoes the above steps, which is done via centrifuge. The broth is then removed and imersedd in a substance that breaks down cell membranes, releasing the insulin from the bacteria.

8. The two protein chains of insulin are brought together via enzymes that ensure the proper amino acids pair with their correct counterparts, and, if done correctly, the disulfide bonds between the two strands form and the insulin is nearly ready for its packaging stage.

9. The final step before the insulin is ready for human consumption, however is crystallization, or "drying." The insulin is mixed with zinc, which helps it to form stable crystals, and dried to powder consistency. This powder can be rehydrated in solution and poured into vials for subcutaneous injection.

This process is so effecient that it takes under 30 minutes for a multi-million colony of E.coli to express human insulin via recombinant DNA technology, thus making synthetic insulin readily available to diabetics worldwide.

References:

American Diabetes Association. "Diabetes Forecast," April 2011. http://www.diabetesforecast.org/2013/jul/making-insulin.html

Cold Spring Harbor Laboratory. "How Insulin is Made." 2010.http://www.dnalc.org/view/15928-How-insulin-is-made-using-bacteria.html

Höppener JW, de Pagter-Holthuizen P, Geurts van Kessel AH, Jansen M, Kittur SD, Antonarakis SE, Lips CJ, Sussenbach JS (1985). "The human gene encoding insulin-like growth factor I is located on chromosome 12". Hum. Genet. 69 (2): 157–60.

Images courtesy of: http://www.nlm.nih.gov/exhibition/fromdnatobeer/exhibition-interactive/recombinant-DNA/recombinant-dna-technology-alternative.html