Group 5 Insulin Production Script

Clarice: Good afternoon everyone, we are the group 5 and today we will be discussing about the insulin
production. My group mate jean will give us an introduction to how insulin is produced.

Jean: Insulin is a hormone created by your pancreas that controls the amount of glucose in your
bloodstream at any given moment. It also helps store glucose in your liver, fat, and muscles. Finally, it
regulates your body’s metabolism of carbohydrates, fats, and proteins. Sound important? That’s
because it is. “Without proper insulin function, your body can’t store glucose in your muscles or liver,
but neither can it make any fat. Instead, the fat breaks down and produces, among other things, keto
acids,” says endocrinologist Irl Hirsh, MD. If the levels of these acids grow too high, the imbalance can
trigger diabetic ketoacidosis, a potentially fatal condition. When you eat, your blood glucose levels rise,
and this leads a typical person’s pancreas to release insulin, so that the sugar can be stored as energy for
later use. Without that pancreatic ability, as a person with either type 1 diabetes or advanced type 2
diabetes, your blood sugar levels may rise dangerously high, or drop too low.

History of Insulin
Yeonah: Before insulin was discovered in 1921, patients with diabetes did not have long lives. The most
successful therapy was to place diabetic patients on stringent carbohydrate-restricted diets. This could
provide patients with a few more years of life, but could not entirely cure them. Patients have died of
hunger as a result of strict diets consisting of only 450 calories per day. Sir Edward Albert Sharpey-Shafer
first proposed in 1910 that patients with diabetes had just one hormone lacking from their pancreas.
Insulin is derived from the Latin word insula, which means "island". Frederick Banting, a young surgeon,
discovered how to extract insulin from a dog's pancreas in 1921. The separated substance appeared to
be "thick brown sludge," but they had no idea it would lead to life and hope for millions of diabetics.

Arhiana: Leonard Thompson, a 14-year-old child dying of diabetes in a Toronto hospital in January 1922,
became the first person to receive an injection of insulin. His critically high blood glucose levels
decreased to near-normal levels within 24 hours. The Nobel Prize for Medicine was awarded to Banting
and Macleod in 1923. Eli Lilly, a pharmaceutical company, began mass-producing insulin shortly after.
Manufacturers produced several slower-acting insulins throughout the decades that followed, with
Novo Nordisk Pharmaceuticals, Inc. introducing the first in 193631. Insulin from cows and pigs was used
to treat diabetes for many years and saved millions of lives, though it was not optimal, as many people
developed allergic responses to it. The invention of DNA cloning by Stanley Cohen and Herbert Boyer
heralded the beginning of genetic engineering, which allowed genes to be easily transferred across
various biological species. Their discovery led to the creation of various recombinant proteins with
medicinal uses, including insulin and growth hormone. In 1978, E. coli bacteria were used to
manufacture the first genetically engineered synthetic “human” insulin. Eli Lilly went on to market the
first commercially accessible biosynthetic human insulin under the brand name Humulin in 1982, which
was authorized by the FDA for medicinal use in humans
The Process of Insulin
Hannah: The first insulin production method that will be discussed here involves the synthesis of
proinsulin. An alternative two-chain method where the A and B chains of insulin are produced
separately can also be used. Recombinant E. coli are used to produce adequate quantities of proinsulin.
This recombinant protein is produced by incorporating proinsulin-producing plasmids into E. coli35. The
transformed cells are then grown on tryptic soy broth containing the antibiotic kanamycin monosulfate.
The plasmid contains a kanamycin monosulfate resistance gene along with the proinsulin coding genes,
so the transformed E. coli can survive in the broth. However, kanamycin monosulfate kills the E. coli cells
that have not been transformed.

Fermentation and Parameters

Clarice: In this method, six 200ml test tubes are used to grow 0.5g of the initial transformed E. coli cells
in 1lL tryptic soy broth solution with 0.5g of kanamycin monosulfate. After the cells have been left to
grow in the medium for 24 hours at 37°C they are inoculated in a bioreactor to promote growth and
production of proinsulin. After 24 hours, the transformed E. coli has consumed and depleted all the
nutrients in the test tubes, hence they are placed in the bioreactor to support further growth. This
bioreactor has a total volume of 23L and a working volume of 16L. 1L of E. coli and depleted growth
medium is taken and mixed with 9L of fresh growth medium in the bioreactor. Now, these cells will
receive carbon from glycerol and yeast, nitrogen from ammonium sulfate and thiamine, and inorganic
nutrients from potassium dihydrogen phosphate and dipotassium phosphate, which also act as buffers
to maintain pH. Trace elements will be provided by sodium citrate, magnesium sulfate, and a vitamin
solution.

Cell Isolation by Centrifugation

Jean: Cell isolation is the first step in down streaming of the insulin made by transformed E. coli cells.
This process is also referred to as cell harvesting because the proinsulin inclusion bodies are harvested
using both filtration and centrifugation.

Cell Lysis by Homogenization

Clarice: The proinsulin inclusion bodies present inside the cell contain insulin precursor products in the
form of proinsulin fusion proteins. Since they are present in dense aggregates, they are protected from
being processed into the soluble form within the cytoplasm.

Inclusion Body Separation by Centrifugation

Yeonah: After the E. coli have been lysed, the inclusion bodies need to be isolated from the cell debris.
For this purpose, centrifugation can be used for reverse osmosis. Since the proinsulin inclusion bodies
are dense, they will sink to the bottom. However, the speed of centrifugation must be higher (15000 x g
for 30 minutes) than before, since the inclusion bodies have a lower density than the intact bacterial
cells. After centrifugation, the supernatant is discarded while proinsulin and some impurities remain in
the tube.
Solubilization of Inclusion

Hannah:Bodies After the separation of inclusion bodies, proinsulin is in an insoluble form and therefore
must be solubilized. This is accomplished through the addition of denaturing agents such as urea or
guanidium hydrochloric acid, which will release the fusion proteins. This process is followed by the
addition of either β-mercaptoethanol or DTT, which are reducing agents, to break the disulfide bonds
present within the proinsulin fusion proteins.

Sulfitolysis Sulfitolysis

Arhiana: first involves breaking the disulfide bonds by adding reducing agents. These bonds get broken
during solubilization or other earlier steps of purification.

Additional Separation

Clarice: Before renaturation, the impurities and reagents from solubilization and sulfitolysis must be
removed via centrifugation at 17700 x g for 33 minutes.

Dialysis

Yeonah: This process is used to remove the previously used denaturants and dissolved reagents without
chemically modifying the fusion protein product.

Renaturation

Jean: The process of renaturation involves the correct folding of proteins, which depends heavily upon
the correct formation of disulfide bonds. There are several methods available. Two commonly used
methods are (1) the use of oxidative buffers such as low molarity Tris-HCl or glycine-sodium hydroxide to
oxidize the reduced proinsulin, or (2) conversion of the proinsulin to the S-hexa-sulfonated form via
sulfothiolysis using sodium sulfite, followed by the addition of redox reagents such as cysteamine, GSH
or cysteine couples.

Volume Reduction

Arhiana: After renaturation, the reagents and buffers that were used need to be removed so that the
proinsulin product can be isolated. This can be done by the add.

Alternative Methods for Insulin Production

Hannah: The second commercial method for insulin production is the two-chain method, in which the A
chain and the B chain of insulin are produced separately and then fused. Here, these 2 polypeptides are
cultured in bacteria in two different fermenters and then purified. The purified A and B chains are then
incubated under oxidizing conditions to form the disulfide bonds that are present in human insulin.

Bio-production Steps

Yeonah: Gene Isolation Complementary DNA (cDNA) molecules encoding chain A and chain B are
obtained from human insulin mRNA using reverse transcription. The cDNAs of both chains are amplified
by PCR.
Insertion into the Plasmid

Arhiana: Two plasmids are cut using restriction enzymes to insert the DNA sequence for the A chain or
the B chain separately. Each chain is extended with an ATG initiation codon on the 5’ terminus to begin
the translation process, while the termination signal is present on the plasmid at 3’end of the restriction
sites. The restriction sites of EcoR1 and BamH1 contain one of the chain genes in both plasmids. The
plasmid also contains a lacZ gene, which encodes for β-galactosidase, allowing for colony screening.
Specific DNA ligases are added to bind the inserted chain gene into the plasmid.

Transfection

Jean: The entry of recombinant plasmids into bacterial cells is called transfection. Different techniques
can be used for the transformation of E. coli, such as treatment with CaCl2 or electroporation
techniques. After the plasmid’s entry, the cells become transformed.

Medium Preparation

Hannah: LB broth is used as a culture medium for E. coli. It is first dissolved and the solution is
autoclaved for sterilization, and then ampicillin and lactose are added. The medium is inoculated with
transformed E. coli cells57. STR bioreactors are used for fermentation of the two E. coli strains encoding
the insulin chains. The bioreactors are sterilized and the pH, pO2 probe, condensers, and air inlet are
calibrated.

Bioreactor Fermentation

Clarice: Shake flasks are used for small-scale fermentation of recombinant E. coli cells encoding the A
and B chains in the enriched medium, which are then used for the large-scale fermentation process. The
ampicillin resistance and lacZ genes present on the plasmids are used for the identification of
successfully transformed cells, as they show resistance against the ampicillin present in the growth
medium and encode β-galactosidase. These successfully transformed cells are retained for replication
under optimal conditions and then transferred into a bioreactor for commercial production. The A and B
chains are synthesized as their respective E. coli strains replicate in separate fermenters.

Crude Product Isolation

Jean: The bacterial cells are removed from the bioreactor tank and must be lysed with one of several
methods, such as enzyme digestion, sonication, or freezing and thawing of cells. The use of lysosome
enzymes is preferred for large-scale operations, as it digests the bacterial outer layer to release the
insulin into the surrounding media, so detergents can later be added to remove the cell wall.

Purification

Yeonah: Cell components are separated from the two desired insulin chains. Gel filtration and ion-
exchange chromatography methods are used to remove impurities.

Insulin Chain Isolation

Clarice: The isolated purified protein has an insulin chain fused with β-galactosidase, as it has been
linked with the gene incorporated into the plasmid and translated along with it. Cyanogen bromide is
used to separate the insulin chains from β-galactosidase, as it splits the protein at the methionine
residue which begins the β-galactosidase protein.

Chain Joining

Hannah: The insulin chains (A and B) are treated with sodium dithionite and sodium sulfite to form the
disulfide bonds that bridge the chains. This whole process is called reduction-reoxidation, and it is
induced by β-mercaptoethanol and air oxidation to synthesize human insulin.

Yeonah: Reverse-Phase High-Performance Liquid Chromatography RP-HPLC is performed to remove the

remaining impurities or reagents. The purified, active human insulin can then be packed and sold by the
industry.

BENEFITS IN PRODUCING INSULIN IN THE BODY

Clarice: -Insulin helps your body to break up the sugar and fats that stuck up in the body and causes
diabetes. Insulin production helps deliver the glucose in the blood to the cells this prevents glucose from
building up in the blood streams

Hannah: -The insulin signals your muscles in breaking down glucose to help stabilized the blood sugar
level and prevent Diabetes

DISADVANTAGES OF NOT PRODUCING INSLIN IN THE BODY

Arhiana: - Without insulin our body is not able to control the sugar in the body resulting obesity or
weight loss that can lead to serious condition called Diabetes (DIABETIC KETOACIDOSIS), that can further
more lead to pancreatic cancer.

Jean: That is all. I hope you learned something from our discussion today, My name is (introduce
yourself). Once again we are the group 5 and thank you for listening.

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