Microbiology in details Latest technological development in field of Biotechnology,new drug molecules ,enzymes ,new drug molecule synthesis methods.Diabetis treatment , hybridoma technique , human insuline

Monday, May 12, 2008

Recombinant DNA Technology in the Synthesis of Human Insulin

Recombinant DNA Technology in the Synthesis of Human Insulin
Since Banting and Best discovered the hormone, insulin in 1921. diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.
Although bovine and porcine insulin are similar to human insulin, their composition is slightly different.Consequently, a number of patients' immune systems produce antibodies against it, thus neutralising its actions and resulting in inflammatory responses at injection sites. Added to these
adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance, as well as a projected decline in the production of animal derived insulin.These factors led researchers to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart.
This has been achieved using Recombinant DNA technology.
This method is a more reliable and sustainable method than extracting and purifying the abattoir by-product.Understanding the genetics involved.
The structure of insulin.
Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains are linked by a disulfide bond.
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Inside the Double Helix.
The genetic code for insulin is found in the DNA at the top of the short arm of the eleventh chromosome. It contains 153 nitrogen bases (63 in the A chain and 90 in the B chain).DNA Deoxyribolnucleic Acid), which makes up the chromosome, consists of two long intertwined helices, constructed from a chain of nucleotides, each composed of a sugar deoxyribose, a phosphate and nitrogen base. There are four different nitrogen bases, adenine, thymine, cytosine and guanine.The synthesis of a particular protein such as insulin is determined by the sequence in which these bases
are repeated .
Insulin synthesis from the genetic code.
The double strand of the eleventh chromosome of DNA divides in two, exposing unpaired nitrogen bases which are specific to insulin production .
The role of the mRNA strand, on which the nitrogen base thymine is replaced by uracil, is to carry genetic information, such as that pertaining to insulin,from the nucleus into the cytoplasm, where it attaches to a ribosome
The nitrogen bases on the mRNA are grouped into threes, known as codons. Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to a specific amino acid, collectively known as an anti-codon pair with complementary bases (the codons) on the mRNA. The Vector (Gram negative E. coli).
A weakened strain of the common bacterium, Escherrichia coli (E. coli), an inhabitant of the human digestive tract, is the 'factory' used in the genetic engineering of insulin. When the bacterium reproduces, the insulin gene is replicated along with the plasmid, a circular section of DNA . E. coli produces enzymes that rapidly degrade foreign proteins such as insulin. By using mutant strains that lack these enzymes, the problem is avoidedIn E. coli, B-galactosidase is the enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the insulin gene needs to be tied to this enzyme. Inside the genetic engineer's toolbox
Restriction enzymes, naturally produced by bacteria, act like biological scalpels, only recognising particular stretches of nucleotides, such as the one that codes for insulin.
This makes it possible to sever certain nitrogen base pairs and remove the section of insulin coding DNA from one organism's chromosome so that it can manufacture insulin . DNA ligase is an enzyme which serves as a genetic glue, welding the sticky ends of exposed nucleotides together.
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Manufacturing Human insulin.

The first step is to chemically synthesise the DNA chains that carry the specific nucleotide sequences characterising the A and B polypeptide chains of insulin The required DNA sequence can be determined because the amino acid compositions of both chains have been charted. Sixty three nucleotides are required for synthesising the A chain and ninety for the B chain, plus a codon at the end of each chain,signalling the termination of protein synthesis. An anti-codon, incorporating the amino acid, methionine, is then placed at the beginning of each chain which allows the removal of the insulin protein from the bacterial cell's amino acids.
The synthetic A and B chain 'genes' are then separately inserted into the gene for a bacterial enzyme, B-galactosidase, which is carried in the vector's plasmid. At this stage, it is crucial to ensure that the codons of the synthetic gene are compatible with those of the B-galactosidase. The recombinant plasmids are then introduced into E. coli cells. Practical use of Recombinant DNA technology in the synthesis of human insulin requires millions of copies of the bacteria whose plasmid
has been combined with the insulin gene in order to yield insulin. The insulin gene is expressed as it replicates with the B-galactosidase in the cell undergoing mitosisThe protein which is formed, consists partly of B-galactosidase, joined to either the A or B chain of insulin . The A and B chains are then extracted from the B-galactosidase fragment and purified. Biological implications of genetically engineered Recombinant human insulin. Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely identical to that of the natural molecule. This reduces the possibility of complications resulting from antibody production. In chemical and pharmacological studies, commercially available Recombinant DNA human insulin has proven indistinguishable from pancreatic human insulin. Initially the major difficulty encountered was the contamination of the final product by the host cells, increasing the risk of contamination in the fermentation broth. This danger was eradicated by the introduction of purification processes. When the final insulin product is subjected to a battery of tests, including the finest radio-immuno assay techniques, no impurities can be detected. The entire procedure is now performed using yeast cells as a growth medium, as they secrete an almost complete human insulin molecule with perfect three dimensional structure.
This minimises the need for complex and costly purification procedures. The issue of hypoglycaemic complications in the administration of human insulin.
Since porcine insulin was phased out, and the majority of insulin dependent patients are now treated with genetically engineered recombinant human insulin, doctors and patients have become concerned about the increase in the number of hypoglycaemic episodes experienced. Although hypoglycaemia can be expected occasionally with any type of insulin, some people with diabetes claim that they are less cognisant of attacks of hypoglycaemia since switching from animal derived insulin to Recombinant DNA human insulin.(16) In a British study,
published in the 'Lancet", hypoglycaemia was induced in patients using either pork or human insulin, The researchers found "no significant difference in the frequency of signs of hypoglycaemia between users of the two different types of insulin."
An anecdotal report from a British patient who had been insulin dependent for thirty years, stated that she began experiencing recurring, unheralded hypoglycaemia only after substituting Recombinant DNA human insulin for animal derived insulin. After switching back to pork insulin to ease her mind, she hadn't experienced any unannounced hypoglycaemia. Eli Lilly and Co., a manufacturer of human insulin, noted that a third of people with diabetes, who have been insulin dependent for over ten years, "lose their hypoglycaemic warning signals, regardless of the type of insulin they are taking." Dr Simon P. Wolff of the University College of London said in an issue of Nature , "As far as I can make out, there's no fault (with the human insulin)." He concluded, "I do think we need to have a study to examine the possible risk.

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