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It is the illness that is occuring when the body tissues are attacked by the body immune system . Immune system is a 'complex organization' within the body designed to seek and destroy the foreign bodies like pathogens. Patients suffering from this condition have unusual antibodies in their blood that targets the body tissues. The presence of one autoimmune disorder can trigger another disorder. This is more frequent in women than in men that may be due to the action of estrogen on immune system.
Diabetes mellitus type 1
This is a form of diabetes mellitus that results from autoimmune destruction of beta cells of the pancreas. The subsequent lack of insulin leads to increased blood and urine glucose. The symptoms are common to that of diabetes polyuria, polydipsia, polyphagia and weight loss results. It is fatal unless treated with insulin. The cause of type 1 diabetes is believed to be of immunological origin. Type 1 can be distinguished from type 2 diabetes via a C-peptide assay, which measures endogenous insulin production.
Type 1 treatment must be continued indefinitely in all cases. It proves to be burdensome in many patients in controlling the level of insulin and this leads to extreme conditions like low blood sugar and high blood sugar. Low blood sugar may lead to seizures or episodes of unconsciousness and requires emergency treatment. High blood sugar may lead to increased tiredness and can also result in long term damage to other organs such as eyes and joints.
Cause of type 1 diabetes
Type 1 diabetes is a polygenic disease, meaning many different genes contribute to its expression. Depending on locus or combination of loci, it can be dominant, recessive, or somewhere in between. The strongest gene, IDDM1, is located in the MHC Class II region on chromosome 6, at staining region 6p21. It is believed to be responsible for the histocompatibility disorder characteristic of type 1: Insulin-producing pancreas cells (beta cells) display improper antigens to T cells. This finally results in the production of antibodies that attack the beta cells. Weaker genes are also located on chromosomes 11 and 18.
Insulin glulisine is a rapid-acting analogue of Insulin that differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid. Chemically, it is 3B-lysine-29B-glutamic acid-human insulin, has the empirical formula C258H384N64O78S6 and a molecular weight of 5823. It works by lowering levels of glucose in the blood.
Mode of Action
Synthetic Insulin acts by binding to the receptor on the cell membrane of the target cells. Insulin receptors are membrane glycoprotein composed of separate insulin-binding (alpha-subunit) and signal transduction (beta-subunit) domains. Binding results in activation of a tyrosine kinase in the beta-subunit that auto-phosphorylates the receptor. The phosphorylated receptor in turn phosphorylates other protein substrates beginning with insulin-receptor substrate (IRS) 1 and 2. The insulin signal is further propagated through a phosphorylation network involving other intracellular substances, leading to the various metabolic actions of insulin. Through activation of these signaling pathways, insulin acts as a powerful regulator of metabolic function. Furthermore, insulin receptor-mediated activation of the mitogen-activated protein (MAP) kinase pathway has been implicated in insulin's effects on growth. Of clinical relevance, defects in insulin signaling have been demonstrated in several of the insulin resistance syndromes.
Production of the insulin glulisine protein.
The production of the protein is to be carried out by recombinant DNA technology in Escherichia coli (E. coli) stain k 12and the protein prodeced differs from human insulin by two amino acid substitutions on the B chain of the protein.This substitution will increase the action rate of the protein than human insulin. This is because Insulins have a pronounced tendency to form hexamers that need to disintegrate to dimers and monomers to be pharmacologically active. The amino acid substitutions in insulin glulisine destabilize the hexamers and therefore enable a faster onset of action than that achieved with human insulin.
The substitution taking place is Asn is replaced by Lys at position 3B and Lys is replaced by Glu at position 29B.
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 (see fig. 3) are linked by a disulfide bond. 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).
Procedure for the production.
Recombinant Dna technique for production biosynthetic insulin:
Recombinant DNA is a form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA (rDNA) is introduced through the addition of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a specific purpose, such as antibiotic resistance. It differs from genetic recombination, in that it does not occur through processes within the cell, but is engineered. A recombinant protein is a protein that is derived from recombinant DNA.
Materials and methods
Restriction enzymes that would cleave the 2 chains of insulin dna
A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. This is referred as Molecular Scissors ass it is widely used in genetic engineering for the insertion of the Foreign gene into an organism's genome. Here we are using PstI or PvuI restriction enzymes.
The Vector pbr322
pBR322 is a plasmid developed form E. coli. pBR322 was the first artificial plasmid to be developed and was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. pBR322 has 4361 base pairs and contains a replicon region (source plasmid pMB1), the ampR gene which encodes the ampicillin resistance protein (source plasmid [RSF2124]) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has unique restriction sites for more than forty restriction enzymes. There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tetR gene. The origin of replication or ori site in this plasmid is pMB1.
The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene. The presence of restriction sites within the markers tetr and ampr permits an easy selection for cells transformed with the recombinant pBR322. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.
Advantages of pbr322
An ideal plasmid vector must have the following functions:
(1) minimum amount of DNA,
(2) relaxed replication control,
(3) at least two selectable markers,
(4) only one (unique) recognition site for at least one restriction endonuclease, and
(5) for easy selection of the recombinant DNA, this unique restriction site must be
We need to take two such vectors and insert chain a in one and the chain b of the insulin gene in the other vector. Using the Pst I or PvuI restriction enzymes cleave the vectors and insert the DNA chains of insulin and after the insertion ligate them by using the DNA ligase.
63 nucleotides are required for synthesising the A chain and ninety for the B chain of the insulin, 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.
Transfection of E coli k12 strain.
Cell transfection, or the introduction of foreign DNA into a cell, can be accomplished chemically, biologically, or mechanically. Here we employ electroporation. Electroporation uses electricity to increase the permeability of the eukaryotic cell membrane, allowing foreign DNA to pass easily inside. This method is good for infecting large numbers of cells at once. It is widely used in molecular biology for the transfection process. The host cell which is used for the transfection process is E.coli k12 strain. Ecoli is a gram negative bacteria which is rod shaped and ahs the size of about 2 µm long and 0.5 µm wide. It divides rapidly by binary fission. It is the first organism whose complete genome is fully understood. K12 strain is widely used in lab. One of the main reasons why this microbe is a key research tool is that it is safe to handle and easy to grow.
The insulin is introduced into an E. coli cell Source: Novo-Nordisk promotional brochure, pg 16 .
Selection of transformed 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 mitosis. Since we used PstI or PvuI restriction enzymes, the transformed cells are not able to grow on amp rich plates but they grow on tet as their amp resistant gene gets disrupted. Thus the transformed cells can be extracted. The transformed bacteria then undergoes a fermentation process. They grow and multiply increasing the yield of protein. After multylpying the cells are taken and the protein is extracted. The recombinant protein 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.
Source: Watson, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 456.
The two chains are mixed and joined in a reaction that forms the disulfide cross bridges, resulting in pure Humulin - synthetic human insulin. It is then purified to obtain pure insulin for clinical uses.
Human insulin molecule. Source: Source: Watson, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 456.
The major problem surfacing is the contamination issues that might be introduced during the fermentation procedures which could include the proteins from host bacterial cells, host DNA , phages . Ecoli has a poor secretion capability and has problems with plasmid instability. Loss of the plasmid DNA after generation creates problem.
Insulin glulisine displays a time-concentration and time-action profile with a more rapid onset, earlier peak effect in lowering blood glucose levels, and a shorter duration of action than the short-acting insulin preparation of regular human insulin. The time-concentration and time-action profiles of insulin glulisine define it as a member of the rapid-acting insulin subfamily of short-acting insulin preparations
Case study about the biosynthetic insulin and its potential market.
Although growth of human insulin sales was initially modest, this product line became the predominant insulin group by the mid-1990s. The market for insulin in the United States is currently growing at more than 10% per year. Between May 2000 and May 2001, it grew at 11% to $1.4 billion. Growth is projected to continue at that rate or more at least until 2020, when insulin sales are projected to exceed $7.5 billion. Much of this growth will be fueled by the introduction of more and more insulin analogs. The data do not account for the potential impact of inhaled insulin, if and when it is released for commercial use.( Aventis: Annual Report, 2001)
Retail price comparison of different forms of injected insulin.