01 Gene Technology for Insulin Production

1. Gene technology for insulin Gene technology for insulin
productionproduction
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2. Steps involved in the genetic engineering of Steps involved in the genetic engineering of
bacteria to synthesise human insulinbacteria to synthesise human 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 bacterial gene are then transferred to the bacterial
cells (transformation)cells (transformation)
•Transformed bacteria are then clonedTransformed bacteria are then cloned
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DNA ligaseDNA ligase

4. 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, 30 of protein. It consists of 51 amino acid, 30 of
which constitute one polypeptide chain, which constitute one polypeptide chain,
and 21 of which comprise a second chain. and 21 of which comprise a second chain.
The two chains are linked by a disulfide The two chains are linked by a disulfide
bond.bond.
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Source: Chance, R. and Frank B. - Research, development,
production and safety of Biosynthetic Human Insulin.

5. •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 of -galactosidase enzyme of
E. coliE. coli
•Methionine triplet code and stop codes are Methionine triplet code and stop codes are
added to the cDNA for each of the insulin added to the cDNA for each of the insulin
genegene
•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 cuts treated with cyanogen bromide which cuts
the amino acid sequence at methioninethe amino acid sequence at methionine
•Insulin forms when the mixture of A and B Insulin forms when the mixture of A and B
chains is treated to promote formation of chains is treated to promote formation of
disulphide bondsdisulphide bonds
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6. •Latest method for manufacturing Latest method for manufacturing
genetically engineered human insulin use genetically engineered human insulin use
eukaryotic yeast cellseukaryotic yeast cells
•Yeast cells can use eukaryotic promoter Yeast cells can use eukaryotic promoter
sequences and have Golgi bodies, so that sequences and have Golgi bodies, so that
they produce insulin that is released they produce insulin that is released
already in the correct 3-dimensional already in the correct 3-dimensional
conformation to achieve maximum activity conformation to achieve maximum activity
in humansin humans
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7. The advantages of treating diabetics with The advantages of treating diabetics with
human insulin produced by gene technologyhuman insulin produced by gene technology
•It is chemically identical to the human insulin, It is chemically identical to the human insulin,
little chance of an immune responselittle 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 responsehuman cell surface membranes, rapid response
•Like natural human insulin, duration of response Like natural human insulin, duration of response
shortershorter
•Overcomes problems related to development of Overcomes problems related to development of
a tolerance to insulin from pigs or cattlea 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 vegetarian cattle insulin, religious objections or vegetarian
objectionsobjections
•Extraction of insulin from pancreases of pigs and Extraction of insulin from pancreases of pigs and
cattle is expensivecattle is expensive
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8. Why promoters need to be transferred Why promoters need to be transferred
along with the desired genesalong with the desired genes
•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 permits information, in the form of DNA sequences, that permits
the proper activation or repression of the gene which it the proper activation or repression of the gene which it
controls, i.e. whether RNA is synthesized or notcontrols, i.e. whether RNA is synthesized 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 promoter transcription factors. These factors bind to the promoter
DNA sequences and the end result is the recruitment of DNA sequences and the end result is the recruitment of
RNA polymerase, the enzyme that synthesizes the RNA RNA polymerase, the enzyme that synthesizes the RNA
from the coding region of the gene. from the coding region of the 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 than Now synthetic DNA can be made rather than rather than
trying to make use of natural promoterstrying to make use of natural promoters
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11. •In eukaryotes, the process is more complicated, In eukaryotes, the process is more complicated,
and at least seven different factors are and at least seven different factors are
necessary for the transcription of an RNA necessary for the transcription of an RNA
polymerase II promoterpolymerase II promoter
•Eukaryote promoters may not have the intended Eukaryote promoters may not have the intended
effect in prokaryotic cellseffect in prokaryotic cells
•When genes are transferred from eukaryotes to When genes are transferred from eukaryotes to
prokaryotes, it is essential that a suitable prokaryotes, it is essential that a suitable
prokaryote promoter is added to the gene before prokaryote promoter is added to the gene before
it forms recombinant DNA with the plasmid it forms recombinant DNA with the plasmid
vectorvector
•If eukaryote promoters are to be transferred with If eukaryote promoters are to be transferred with
eukaryotic genes, into eukaryotic cells of a eukaryotic genes, into eukaryotic cells of a
different species, then care must be taken to different species, then care must be taken to
ensure that all of the relevant code is included ensure that all of the relevant code is included
(TATA box and E box)(TATA box and E box)
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