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Human Insulin and Recombinant DNA TechnologyBY: Nidhi Uppangala | Category: Others | Submitted: 2010-06-01 19:47:22
Article Summary: "Impaired insulin production in the beta cells of pancreas leads to the condition known as Diabetes Miletus. To treat the diabetic patient researchers produced Humulin using recombinant DNA technology by inserting human insulin gene into a vector (E. coli)..."
Impaired insulin production in the beta cells of pancreas leads to the condition known as Diabetes Miletus. The bovine and porcine insulin are used to treat Patients with diabetes mellitus. But the composition of bovine and porcine insulin are slightly different from the human insulin, consequently the patient's immune system produced antibodies against animal origin insulin and also these antibodies induced inflammatory response at injection sites. Antibodies also neutralised the biological action of animal origin insulin. To overcome all these problems researchers produced Humulin using recombinant DNA technology by inserting human insulin gene into a vector (E. coli).
Humulin Production Method:
1. DNA coding for A and B polypeptide chains of insulin are chemically synthesised a in the lab. Sixty three nucleotides are sequenced to produce A chain of insulin and ninety nucleotide long DNA designed to produce B chain of insulin, plus terminator codon is added at the end of each chain sequence. Anti-codon for methionine is added at the beginning of the sequence to distinguish humulin from the other bacterial proteins.
2. Chemically synthesized A and B chain DNA sequence are inserted into one of the marker gene which are present in the plasmid vector. Genes are inserted into the plasmid with the help of enzymes known as endonuclease and ligase.
3. The vector plasmids with the insulin gene are then introduced into the E. coli bacterial cell. These cells are then allowed to replicate by mitosis, along with the bacterial cell recombinant plasmid also gets replicated producing the human insulin.
4. A and B polypeptide chains of insulin are then extracted and purified from the fomenters in the lab. High-Performance Liquid Chromatography (HPLC) is used to get 100% pure humulin from the mixture of proteins.
5. The A and B polypeptide chains of insulin are mixed together and connected with each other by disulphide bond, forming the Humulin or synthetic human insulin
Advantages of Humulin:
Humulin is the one and only human protein produced in the bacteria with identical chemical structure to that of the natural human insulin. Administration of humulin reduces the possibility of antibody production and inflammatory response in diabetic patients. Major difficulty is the extraction of humulin from a mixture of host proteins present in the fermentation broth.
Now days to overcome this extraction problem synthetic human insulin are produced in the yeast cell instead of E. coli using the same procedure. As yeast is Eukaryotes they secrete the whole humulin molecule with perfect three dimensional structures, reducing the need for complex and time consuming purification methods.
Now most of the diabetic patients are treated with synthetic human insulin. Small group of patients claim that episodes of hyperglycaemic complications have been increased after shifting from animal origin insulin to humulin. No study till date shows the difference between the frequency of hyperglycaemic complications in patient using humulin (synthetic human insulin) and animal origin insulin.
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