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Production of Insulin using rDNA Technology


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I write this article to tell people about the principle behind the production of insulin from rDNA technology. This technology is being used to produce a variety of substances today, and insulin was the first among them.



Recombinant DNA Technology


The need for various biochemical substances to treat diseases and for therapy is increasing by the day. The biological synthesis, rather than chemical manufacture, has gained attention because biological synthesis is a natural process, and hence, chances of rejection by the body are less. This biological synthesis has been made a lot easier by recombinant DNA technology. Recombinant DNA technology is nothing but the insertion of a foreign DNA material into the host body so that the host can produce the protein of interest.
The development of recombinant DNA technology quickly led to using bacteria producing important medicinal products such as therapeutic proteins. Insulin was the first recombinant protein that was approved by the FDA for use in humans. The insulin hormone was first synthesized in 1982 in the Escherichia coli bacteria and since then, the world has seen a revolution in the production of recombinant products.

Insulin


Insulin is a hormone produced by cells in the pancreas called beta cells which are present in the islets of Langerhans. When insulin is released into the bloodstream, it plays a vital role in carbohydrate metabolism by converting glucose to glycogen, thereby stimulating the uptake of glucose by muscle cells, where it can be broken down to synthesis ATP molecules. Type-1 or insulin dependent Diabetes mellitus is caused by an inadequate production of insulin by the beta cells. This decreased production of insulin results in elevated blood glucose levels and this can cause a number of problems such as high blood pressure, reduced circulation, cataracts and nerve damage. Type-1 Diabetes requires regular injections of insulin to control the blood glucose levels.

Prior to the use of recombinant DNA technology, insulin used to treat Diabetes mellitus was purified from the pancreases of pigs and cows before being injected into diabetics. The purification process was cumbersome, expensive, and often produced impure batches of insulin, Also, purified insulin was ineffective in some individuals and many others developed allergies to insulin produced from cows. In1978, the gene coding for the insulin protein was isolated, cloned into an expression vector plasmid, expressed in bacterial cells and isolated by scientists working at Genentech, a biotechnology company in San Francisco, California. In 1982, this recombinant human insulin was approved by the United States Food & Drug Administration to be marketed under the trade name of Humulin.

Usage of rDNA technology to synthesize Insulin


Bacteria do not normally make insulin. Their mechanism for carbohydrate metabolism is entirely different from that of humans, as they make use of enzymes like amylases to metabolize carbohydrates. Thus, producing insulin in bacterial cells was a significant advance in biotechnology. It remains an outstanding example of the excellent use of microbial biotechnology in action. The human insulin consists of two polypeptide chains called chain A and chain B. Chain A has 21 amino acid residues and chain B has 30 amino acid residues. These two chains are linked to each other by the presence of disulphide bonds between the cystine residues, to create the active hormone. In the pancreas, beta cells secrete both the chains as one single polypeptide unit that is secreted, and then enzymatically cleaved and folded to join the two subunits. When the human genes for insulin were cloned and expressed in bacteria, genes for each of the sub-units were cloned into separate expression vector plasmids containing the lac z gene encoding the enzyme ß-galactosidase and then used to transform the bacteria.

Since the insulin gene is connected to the lac z gene, when bacteria synthesize proteins from these plasmids, they produce a protein that contains ß-galactosidase attached to the human insulin protein to create a ß-gal-insulin fusion protein. This enables a scientist to purify a protein of interest such as insulin. Bacterial colonies were then passed over an affinity column to isolate the ß-gal-insulin fusion protein. The fusion protein was then chemically treated to cleave off the ß-gal, so as to release the insulin protein. Then, purified A and B chains of insulin were mixed together under conditions that allow the two subunits to bind and form the active hormone. After further purification to conform to the FDA guidelines, the recombinant insulin protein was released for patient use as an injectible drug.

Other recombinant proteins


Shortly after insulin became available, growth hormone-used to treat children who suffer from a form of dwarfism-was cloned in bacteria and became available for human use. A short time later, a wide variety of other medicinally important proteins that were once difficult to obtain became readily available as a result of recombinant DNA technology and expressing proteins in bacteria. Some of these therapeutic proteins with valuable applications for treating medical illness in humans that have been expressed and isolated from bacteria are DNase (DNA digesting enzyme), Erythropoietin (protein that stimulates production of RBCs), Blood clotting factor VIII responsible for hemophilia, superoxide dismutase enzyme, etc to name a few.

rDNA Technology: The answer to all major diseases


The introduction of recombinant DNA technology has thus, drastically reduced the cost of drugs and proteins that are otherwise extremely expensive to produce and purify. The usage of bacteria to produce these products is thus an extremely beneficial application of microbial biotechnology. This technology is progressing by the day and bacteria are today being used to produce a wide variety of substances including vaccines. We can only hope that this technology provides the answer to some of the deadly diseases that are threatening mankind today.


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Responses to "Production of Insulin using rDNA Technology"
Guest Author: ram     28 Sep 2012
I like that, nice information is given.


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