Gene technology for insulin Gene technology for insulin productionproduction

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Steps involved in the genetic Steps involved in the genetic engineering of bacteria to synthesise engineering of bacteria to synthesise

human insulinhuman insulin• Identifying and isolating human insulin Identifying and isolating human insulin

gene (cDNA, synthetic DNA or probe)gene (cDNA, synthetic DNA or probe)• cDNA insulin genes cut with cDNA insulin genes cut with restriction restriction

enzymesenzymes (restriction endonucleases) (restriction endonucleases)• Gene transferred to a bacterial plasmidGene transferred to a bacterial plasmid• Plasmid containing the human insulin Plasmid containing the human insulin

gene are then transferred to the gene are then transferred to the bacterial cells (transformation)bacterial cells (transformation)

• Transformed bacteria are then clonedTransformed bacteria are then cloned

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DNA ligaseDNA ligase

The structure of insulinThe structure of insulin

• Chemically, insulin is a small, simple Chemically, insulin is a small, simple protein. It consists of 51 amino acid, protein. It consists of 51 amino acid, 30 of which constitute one 30 of which constitute one polypeptide chain, and 21 of which polypeptide chain, and 21 of which comprise a second chain. The two comprise a second chain. The two chains are linked by a disulfide bond.chains are linked by a disulfide bond.

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Source: Chance, R. and Frank B. - Research, development, production and safety of Biosynthetic Human Insulin.

• The two genes were added into the lac The two genes were added into the lac operon of the operon of the ββ-galactosidase enzyme -galactosidase enzyme of of E. coliE. coli

• Methionine triplet code and stop codes Methionine triplet code and stop codes are added to the cDNA for each of the are added to the cDNA for each of the insulin geneinsulin gene

• E. coliE. coli grown in the presence of lactose grown in the presence of lactose• Proteins separated from bacteria were Proteins separated from bacteria were

treated with cyanogen bromide which treated with cyanogen bromide which cuts the amino acid sequence at cuts the amino acid sequence at methioninemethionine

• Insulin forms when the mixture of A and Insulin forms when the mixture of A and B chains is treated to promote B chains is treated to promote formation of disulphide bondsformation of disulphide bonds

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• Latest method for manufacturing Latest method for manufacturing genetically engineered human insulin genetically engineered human insulin use eukaryotic yeast cellsuse eukaryotic yeast cells

• Yeast cells can use eukaryotic Yeast cells can use eukaryotic promoter sequences and have Golgi promoter sequences and have Golgi bodies, so that they produce insulin bodies, so that they produce insulin that is released already in the correct that is released already in the correct 3-dimensional conformation to 3-dimensional conformation to achieve maximum activity in humansachieve maximum activity in humans

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The advantages of treating diabetics The advantages of treating diabetics with human insulin produced by gene with human insulin produced by gene

technologytechnology• It is chemically identical to the human It is chemically identical to the human

insulin, little chance of an immune responseinsulin, little chance of an immune response• An exact fit in the human insulin receptors in An exact fit in the human insulin receptors in

human cell surface membranes, rapid human cell surface membranes, rapid responseresponse

• Like natural human insulin, duration of Like natural human insulin, duration of response shorterresponse shorter

• Overcomes problems related to development Overcomes problems related to development of a tolerance to insulin from pigs or cattleof a tolerance to insulin from pigs or cattle

• Avoids ethical issues from the use of pig and Avoids ethical issues from the use of pig and cattle insulin, religious objections or cattle insulin, religious objections or vegetarian objectionsvegetarian objections

• Extraction of insulin from pancreases of pigs Extraction of insulin from pancreases of pigs and cattle is expensiveand cattle is expensive

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Why promoters need to be Why promoters need to be transferred along with the desired transferred along with the desired

genesgenes• A A promoterpromoter is a DNA sequence that contains the is a DNA sequence that contains the

information, in the form of DNA sequences, that information, in the form of DNA sequences, that permits the proper activation or repression of the permits the proper activation or repression of the gene which it controls, i.e. whether RNA is gene which it controls, i.e. whether RNA is synthesized or notsynthesized or not

• The promoter contains specific sequences (TATAAT or The promoter contains specific sequences (TATAAT or TTGACA) that are recognized by proteins known as TTGACA) that are recognized by proteins known as transcription factors. These factors bind to the transcription factors. These factors bind to the promoter DNA sequences and the end result is the promoter DNA sequences and the end result is the recruitment of RNA polymerase, the enzyme that recruitment of RNA polymerase, the enzyme that synthesizes the RNA from the coding region of the synthesizes the RNA from the coding region of the gene. gene.

• In prokaryotes, the promoter is recognized by RNA In prokaryotes, the promoter is recognized by RNA polymerase and an associated sigma factor, which in polymerase and an associated sigma factor, which in turn are brought to the promoter DNA by an activator turn are brought to the promoter DNA by an activator protein binding to its own DNA sequence nearbyprotein binding to its own DNA sequence nearby

• Now synthetic DNA can be made rather than rather Now synthetic DNA can be made rather than rather than trying to make use of natural promotersthan trying to make use of natural promoters

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• In eukaryotes, the process is more In eukaryotes, the process is more complicated, and at least seven different complicated, and at least seven different factors are necessary for the transcription factors are necessary for the transcription of an RNA polymerase II promoterof an RNA polymerase II promoter

• Eukaryote promoters may not have the Eukaryote promoters may not have the intended effect in prokaryotic cellsintended effect in prokaryotic cells

• When genes are transferred from When genes are transferred from eukaryotes to prokaryotes, it is essential eukaryotes to prokaryotes, it is essential that a suitable prokaryote promoter is that a suitable prokaryote promoter is added to the gene before it forms added to the gene before it forms recombinant DNA with the plasmid vectorrecombinant DNA with the plasmid vector

• If eukaryote promoters are to be If eukaryote promoters are to be transferred with eukaryotic genes, into transferred with eukaryotic genes, into eukaryotic cells of a different species, then eukaryotic cells of a different species, then care must be taken to ensure that all of care must be taken to ensure that all of the relevant code is included (TATA box the relevant code is included (TATA box and E box)and E box)

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