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					Recombinant DNA Technology in the Synthesis of Human Insulin




      Recombinant DNA Technology in the Synthesis of
                    Human Insulin




Introduction:-

What is Insulin?
       Insulin is actually a hormone in the human body that controls the level of blood
sugar. It is produced by the pancreas and plays a very important role in the conversion of
sugar, or more appropriately, glucose. With the help of insulin, organs like liver, muscles
and fat tissues, absorb glucose from the blood, convert it into glycogen and store it in the
body. The fluctuating levels of this hormone are the major cause of Diabetes mellitus, a
syndrome that is commonly referred to as diabetes. Insulin is injected to into the body to
keep the level of glucose under control.

Structure of Insulin: -

       Insulin is a rather small protein, with a molecular weight of about 6000 Daltons. It
is composed of two chains held together by disulfide bonds. The figure to the right shows a
molecular model of bovine insulin, with the A chain colored blue and the larger B chain




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Recombinant DNA Technology in the Synthesis of Human Insulin


green. You can get a better appreciation for the structure of insulin by manipulating such a
model yourself.

        The amino acid sequence is highly conserved among vertebrates, and insulin from
one mammal almost certainly is biologically active in another. Even today, many diabetic
patients are treated with insulin extracted from pig pancreas.




                  The structure of insulin: The left side is a space-filling model of the insulin monomer, believed to be
        biologically active. Carbon is green, hydrogen white, oxygen red, and nitrogen blue. On the right side is a
        cartoon of the insulin hexamer, believed to be the stored form. A monomer unit is highlighted with the A chain
        in blue and the B chain in cyan. Yellow denotes disulfide bonds, and magenta spheres are zinc ions




Biosynthesis of Insulin: -

        Insulin is synthesized in significant
quantities only in beta cells in the pancreas.
The insulin mRNA is translated as a single
chain precursor called preproinsulin, and
removal of its signal peptide during insertion
into the endoplasmic reticulum generates
proinsulin. Proinsulin consists of three domains:
an amino-terminal B chain, a carboxy-terminal A
chain and a connecting peptide in the middle
known as the C peptide. Within the endoplasmic
reticulum, proinsulin is exposed to several




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Recombinant DNA Technology in the Synthesis of Human Insulin


specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin.
Insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the
cytoplasm. When the beta cell is appropriately stimulated, insulin is secreted from the cell by exocytosis
and diffuses into islet capillary blood. C peptide is also secreted into blood, but has no known biological
activity.


Release of insulin: -

    Beta cells in the islets of Langerhans release insulin in two phases. The first phase
insulin release is rapidly triggered in response to increased blood glucose levels. The
second phase is a sustained, slow release of newly formed vesicles that are triggered
independently of sugar. The description of first phase release is as follows:

     Glucose enters the beta cells through the glucose transporter GLUT2

     Glucose goes into glycolysis and the respiratory cycle where multiple high-energy

            ATP molecules are produced by oxidation
     Dependent on ATP levels, and hence blood glucose levels, the ATP-controlled

            potassium channels (K+) close and the cell membrane depolarizes
                                                                                 2+
     On depolarization, voltage controlled calcium channels (Ca ) open and calcium

            flows into the cells
     An increased calcium level causes activation of phospholipase C, which cleaves the

            membrane phospholipid phosphatidyl inositol 4,5-bisphosphate into inositol 1,4,5-
            triphosphate and diacylglycerol.
     Inositol 1,4,5-triphosphate (IP3) binds to receptor proteins in the membrane of

            endoplasmic reticulum (ER). This allows the release of Ca2+ from the ER via IP3
            gated channels, and further raises the cell concentration of calcium.
     Significantly increased amounts of calcium in the cells causes release of previously

            synthesized insulin, which has been stored in secretory vesicles

    This is the main mechanism for release of insulin. In addition some insulin release takes
place generally with food intake, not just glucose or carbohydrate intake, and the beta cells
are also somewhat influenced by the autonomic nervous system. The signaling mechanisms
controlling these linkages are not fully understood.


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Recombinant DNA Technology in the Synthesis of Human Insulin


       Other substances known to stimulate insulin release include amino acids from
ingested proteins, acetylcholine, released from vagus nerve endings (parasympathetic
nervous system), cholecystokinin, released by enteroendocrine cells of intestinal mucosa
and glucose-dependent insulinotropic peptide (GIP). Three amino acids (alanine, glycine
and arginine) act similarly to glucose by altering the beta cell's membrane potential.
Acetylcholine triggers insulin release through phospholipase C, while the last acts through
the mechanism of adenylate cyclase. The sympathetic nervous system (via Alpha2 -
adrenergic stimulation as demonstrated by the agonists clonidine or methyldopa) inhibit the
release of insulin. However, it is worth noting that circulating adrenaline will activate
Beta2 -Receptors on the Beta cells in the pancreatic Islets to promote insulin release. This is
important since muscle cannot benefit from the raised blood sugar resulting from
adrenergic stimulation (increased gluconeogenesis and glycogenolysis from the low blood
insulin: glucagon state) unless insulin is present to allow for GLUT-4 translocation in the
tissue. Therefore, beginning with direct innervation, norepinephrine inhibits insulin release
via alpha2 -receptors, then subsequently, circulating adrenaline from the adrenal medulla
will stimulate beta2 -receptors thereby promoting insulin release.

       When the glucose level comes down to the usual physiologic value, insulin release
from the beta cells slows or stops. If blood glucose levels drop lower than this, especially to
dangerously low levels, release of hyperglycemic hormones (most prominently glucagon
from Islet of Langerhans' alpha cells) forces release of glucose into the blood from cellular
stores, primarily liver cell stores of glycogen. By increasing blood glucose, the
hyperglycemic hormones prevent or correct life-threatening hypoglycemia. Release of
insulin is strongly inhibited by the stress hormone norepinephrine (noradrenaline), which
leads to increased blood glucose levels during stress.

       Evidence of impaired first phase insulin release can be seen in the glucose tolerance
test, demonstrated by a substantially elevated blood glucose level at 30 minutes, a marked
drop by 60 minutes, and a steady climb back to baseline levels over the following hourly
time points.




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Recombinant DNA Technology in the Synthesis of Human Insulin


Control of Insulin Secretion: -

   Insulin is secreted in primarily in response to elevated blood concentrations of glucose.
This makes sense because insulin is "in charge" of facilitating glucose entry into cells.
Some neural stimuli (e.g. sight and taste of food) and increased blood concentrations of
other fuel molecules, including amino acids and fatty acids, also promote insulin secretion.

   Our understandings of the mechanisms behind insulin secretion remain somewhat
fragmentary. Nonetheless, certain features of this process have been clearly and repeatedly
demonstrated, yielding the following model:

    Glucose is transported into the beta cell by facilitated diffusion through a glucose

       transporter; elevated concentrations of glucose in extracellular fluid lead to elevated
       concentrations of glucose within the beta cell.
    Elevated concentrations of glucose within the beta cell ultimately lead to membrane

       depolarization and an influx of extracellular calcium. The resulting increase in
       intracellular calcium is thought to be one of the primary triggers for exocytosis of
       insulin-containing secretory granules. The mechanisms by which elevated glucose
       levels within the beta cell cause depolarization is not clearly established, but seems
       to result from metabolism of glucose and other fuel molecules within the cell,
       perhaps sensed as an alteration of ATP:ADP ratio and transduced into alterations in
       membrane conductance.
    Increased levels of glucose within beta cells also appear to activate calcium-

       independent pathways that participate in insulin secretion.

   Stimulation of insulin release is readily observed in whole animals or people. The
normal fasting blood glucose concentration in humans and most mammals is 80 to 90 mg
per 100 ml, associated with very low levels of insulin secretion.

   The figure to the right depicts the effects on insulin secretion when enough glucose is
infused to maintain blood levels two to three times the fasting level for an hour. Almost
immediately after the infusion begins, plasma insulin levels increase dramatically. This
initial increase is due to secretion of preformed insulin, which is soon significantly

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Recombinant DNA Technology in the Synthesis of Human Insulin


depleted. The secondary rise in insulin reflects the considerable amount of newly
synthesized insulin that is released immediately. Clearly, elevated glucose not only
simulates insulin secretion, but also transcription of the insulin gene and translation of its
mRNA.




Regulation of Insulin Secretion: -

       Beta cells are unique endocrine cells. They respond positively, in terms of insulin
secretion, not only to changes in the extracellular glucose concentration, but also to
activators of the phospholipase C (cholecystokinin or acetylcholine), and to activators of
adenylate cyclase (glucagon, glucagon-like peptide-1, or gastric inhibitory polypeptide).
Major messengers which mediate glucose action for insulin release are Ca2+, adenosine
triphosphate (ATP) and diacylglycerol (DAG).




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Recombinant DNA Technology in the Synthesis of Human Insulin


Oscillations: -
       Even during digestion, generally one or two hours following a meal, insulin release
from pancreas is not continuous, but oscillates with a period of 3–6 minutes, changing from
generating a blood insulin concentration more than ~800 pmol/l to less than 100 pmol/l.
This is thought to avoid down regulation of insulin receptors in target cells and to assist the
liver in extracting insulin from the blood. This oscillation is important to consider when
administering insulin-stimulating medication, since it is the oscillating blood concentration
of insulin release which should, ideally, be achieved, not a constant high concentration.




                  Fig.: - Insulin release from pancreas oscillates with a period of 3–6 minutes.


        It is also important to consider in that all methods of insulin replacement can never
hope to replicate this delivery mechanism precisely. This may be achieved by delivering
insulin rhythmically to the portal vein or by islet cell transplantation to the liver. Future
insulin pumps hope to address this characteristic.


History of Insulin: -
       Paul Langerhans was a medical student of Berlin University, who discovered a
clump of unidentified tissues in 1869, while observing the pancreas. These tissues were
later named as Islets of Langerhans, by Edouard Laguesse, a French pathologist. Experts at
that time explained that these clump of tissues played an active role in digestion. In the year
1889, two physicians, Oscar Minkowski and Joseph von Mering, took up research on the
basis of Paul Langerhans' discovery. For the purpose of their research they removed the
pancreas from a healthy dog. A few days after the pancreas had been removed, the animal
keeper observed a swarm of flies near the dog's urine. The presence of flies led to the
discovery of sugar in the urine. This relation between blood sugar and pancreas was


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Recombinant DNA Technology in the Synthesis of Human Insulin


established by Oscar Minkowski and Joseph von Mering after further studies.
In the year 1901, Eugene Opie undertook further research and discovered Diabetes
mellitus, a syndrome caused when the Islets of Langerhans were destroyed, either partially
or permanently. In the early 1900's, the phenomenon of diabetes was considered as silent
death. Many scientists and physicians made attempts to develop Islets of Langerhans
artificially as treatment for low levels of insulin and diabetes. Some noted breakthroughs
were achieved by George Ludwig Zuelzer in 1906, E.L. Scott in 1912, and Israel Kleiner in
1919. Nicolae Paulescu, a Romanian medicine professor, isolated these islets for the first
time in a form that was initially known as Pancrein. The first injection of insulin was
developed by a Canadian scientist, Frederick Banting. After many years of research and
considerable help from Charles Best and James Collip, the team of three managed to isolate
islets in the form of insulin. The injection was administered to Leonard Thompson, a 14
year old diabetic. The first dose that was administered on 11th of January 1992 was impure
and produced an allergic reaction. After a further 12 hard working days and nights, the
team came up with a refined and pure dose that produced miraculous results, without any
side effects. Shortly afterwards, a pharmaceutical company, 'Eli Lilly and Company', along
with the scientists, produced an improvised version of insulin that could be commercially
sold and administered. This insulin was, however, sourced from animals. 'Genentech',
another company, was successful in producing biosynthetic human insulin in 1977. In
1982, Genentech and Eli Lilly partnered the first production of biosynthetic human insulin.
         Fredric Banting and Charles Best were awarded the Nobel Prize in 1923 for their
outstanding efforts. Newspaper extracts about the discovery described a scene in the
hospital with the lines: "Grieving family members were often in attendance, awaiting death.
In one of medicine's more dramatic moments the team went from bed to bed, injecting an
entire ward with the new purified extract. Before they had reached the last dying child, the
first few were awakening from their coma, to the joyous exclamations of their families".
Their work was highly recognized but some names of scientists, like Nicolae Paulescu,
have been lost in the pages of history. This wondrous discovery has helped save an
umpteen number of patients suffering from diabetes, from consequences ranging even upto
death.



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Recombinant DNA Technology in the Synthesis of Human Insulin


Background: -
       Diabetes Mellitus is a disease that affects millions of Indians everyday.
       Despite the fact that this affliction is incurable, it is still manageable. Diabetes is a
disorder of metabolism caused by the body producing no or little insulin. There are three
main types of diabetes: type I, type II, and gestational. Type I diabetes is an autoimmune
disease when the body attacks its’ beta cells. It is most likely caused by genetics and
environmental factors. Type II diabetes is when the pancreas stops producing enough
insulin for the body or if the cells in the body stop reacting to insulin. Most likely causes of
Type II diabetes are obesity, family history, high blood pressure, physical inactivity, family
history, and high cholesterol. Gestational diabetes occurs only during pregnancy. Because
of the developing fetus, certain hormones are produced by the body and these hormones
cause the cells of the body to stop recognizing insulin. It can be caused by obesity, family
history, pregnancy at an older age, and previous history of diabetes.
       Diabetes occurs when the pancreas stops producing insulin or when the cells of
ones body stop recognizing insulin. Insulin is a hormone that regulates the metabolism of
glucose, which is the body’s main source of energy. Glucose is a monosaccharide that
starts the process of cellular respiration in eukaryotes. When food is digested, it is broken
down into simple sugars, which are glucose, fructose, and galactose. Without glucose, one
could not live. Insulin is the key that helps the body to use glucose. It is produced in the
pancreas, or more specifically, the islets of Langerhans. These are groups of beta cells that
produce different hormones for the body and are located among the acinar cells of the
pancreas. The pancreas as a whole produces digestive enzymes.
       Insulin is a polypeptide hormone that regulates metabolism in the body. It is
compromised of 51 amino acids, which are split into two polypeptide chains. One chain
contains 30 amino acids and the other 21. These two chains are connected by a disulfide
bond. After a meal is eaten and glucose is released into the bloodstream then the pancreas
releases insulin according to how much glucose was consumed. The insulin then attaches to
the receptors on the cell wall which allow the certain transport molecules on the cell to
allow glucose into the cell. Once glucose is inside of the cell it is broken down in
glycolysis, which is a process where the energy in the bonds in a glucose molecule is



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Recombinant DNA Technology in the Synthesis of Human Insulin


transferred to the phosphate bonds in ATP. ATP is later broken down and the energy
released from the bonds is used by the cell.
        The first type of insulin to be used by humans to treat diabetes was animal insulin,
but synthetic human insulin is now more commonly used.
It is has almost completely replaced animal insulin because synthetic human insulin is
absorbed more quickly into the bloodstream, it is much less likely to cause an allergic
reaction, it is less apt to contain impurities, it is more effective, and it is cheaper to produce.
Despite the fact that synthetic human insulin is almost exact to natural human insulin, some
people find that it is more difficult to foresee hypoglycemia because human insulin makes
it harder for them to be aware of the symptoms.
        There are four main types of human insulin. The types vary according to onset,
peak time, and duration. Onset is the amount of time that it takes for the insulin to start
working, peak time is the time when the insulin is working the best, and duration is the
amount of time that the insulin continues to work. The four types of insulin are: rapid
acting, regular, intermediate acting, and long acting (see figure 1). Different types of
insulin can also be mixed to fit the needs of the individual patient .
        Diabetes is fatal because of several different reasons. The most immediate is that
glucose begins to build up in the blood stream. After the blood glucose level exceeds 180
mg/100 ml, the glucose begins to pass into the urine. Because the body cannot pass glucose
out of the body unless it is suspended in a solution, water is being urinated out constantly
causing dehydration and constant urination, or polyuria. Since glucose is the cells main
source of energy, when it becomes unavailable, the body turns to fat as the main source of
energy. This would not be harmful except when fat is metabolized rapidly and excessively,
ketones begin to appear. Ketones are products of excessive fat metabolism and as they
build up they begin to acidify the blood. The body not only turns to fat as a source of
energy, but to proteins as well. The body begins to break down proteins in order to obtain
glucose from the amino acids and this will result in a lack of tissue protein. Eventually, the
buildup of ketones and acid in the bloodstream will result in a coma and ultimately, death.
        Even when diabetes is treated with regular medication or insulin injections, the
disease has many long term effects. These include heart disease, stroke, nerve damage,
problems in the feet and legs, kidney disease, eye problems, gum disease, respiratory
infections, urinary tract infections, scaly or hard skin, celiac disease, increased risk of colon
and rectal cancer, and hearing loss. Most of these problems are avoidable with extreme care
and many of them are on their way to being cured, but for right now many of these long
term effects are fatal.

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Recombinant DNA Technology in the Synthesis of Human Insulin




                         Insulin types and actions



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Recombinant DNA Technology in the Synthesis of Human Insulin

 Recombinant DNA (rDNA): -
         rDNA stands for recombinant DNA. Before
 we get to the "r" part, we need to understand DNA.
 Those of you with a background in biology
 probably know about DNA, but a lot of ChemE's
 haven't seen DNA since high school biology. DNA
 is the keeper of the all the information needed to
 recreate an organism. All DNA is made up of a base
 consisting of sugar, phosphate and one nitrogen
 base. There are four nitrogen bases, adenine (A),
 thymine (T), guanine (G), and cytosine (C). The
 nitrogen bases are found in pairs, with A & T and G
 & C paired together. The sequence of the nitrogen

bases can be arranged in an infinite ways, and their structure is known as the famous
"double helix" which is shown in the image.
        The sugar used in DNA is deoxyribose. The four nitrogen bases are the same for all
organisms. The sequence and number of bases is what creates diversity. DNA does not
actually make the organism, it only makes proteins. The DNA is transcribed into mRNA
and mRNA is translated into protein, and the protein then forms the organism. By
changing the DNA sequence, the way in which the protein is formed changes. This leads
to either a different protein, or an inactive protein.
        Now that we know what DNA is, this is where the recombinant comes in.
        Recombinant DNA is the general name for taking a piece of one DNA, and
combining it with another strand of DNA. Thus, the name recombinant!
        Recombinant DNA is also sometimes referred to as "chimera". By combining two
or more different strands of DNA, scientists are able to create a new strand of DNA. The
most common recombinant process involves combining the DNA of two different
organisms.




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Recombinant DNA Technology in the Synthesis of Human Insulin


Importance of Recombinant DNA: -
   Recombinant DNA has been gaining in importance over the last few years, and
recombinant DNA will only become more important in the 21st century as genetic diseases
become more prevalent and agricultural area is reduced. Below are some of the areas
where Recombinant DNA will have an impact.
    Better Crops (drought & heat resistance)
    Recombinant Vaccines (i.e. Hepatitis B)
    Prevention and cure of sickle cell anemia
    Prevention and cure of cystic fibrosis
    Production of clotting factors
    Production of insulin
    Production of recombinant pharmaceuticals
    Plants that produce their own insecticides
    Germ line and somatic gene therapy


Production of insulin: -

       One of the biggest breakthroughs in recombinant DNA technology happened in the
manufacture of biosynthetic "human" insulin, which was the first medicine made via
recombinant DNA technology.
       It was the ideal component because it is a relatively simple protein and was
therefore relatively easy to copy, it was so widely used that if researchers could prove that
biosynthetic "human" insulin was safe and effective. Then the technology would be
accepted as such, and it would open the flood gates for many other products to be made this
way.
The specific gene sequence, or oligonucleotide, those codes for insulin production in
human was introduced to a sample colony of E. coli. Only about 1 out of 10 6 bacteria picks
up the sequence. However, this is not really a problem, because the lifecycle is only about
30 minutes for E. coli. This means that in a 24-hour period, there may be billions of E. coli
that are coded with the DNA sequences needed to induce insulin production.



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Recombinant DNA Technology in the Synthesis of Human Insulin


Synthesis of Human Insulin by Recombinant DNA Technology
   a) The nature and purpose of synthesizing human insulin: -
       Since Banting and Best discovered the hormone, insulin in 1921. Diabetic patients,
whose elevated sugar levels (see fig. 1) are due to impaired insulin production, have been
treated with insulin derived from the pancreas glands of abattoir animals. The hormone,
produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the
use and storage of food, particularly carbohydrates.




     Fig. 1: Fluctuations in diabetic person's blood glucose levels, compared with healthy individuals.



Although bovine and porcine insulin are similar to human insulin, their composition is
slightly different. Consequently, a number of patients' immune systems produce antibodies
against it, neutralising its actions and resulting in inflammatory responses at injection sites.
Added to these adverse effects of bovine and porcine insulin, were fears of long term

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Recombinant DNA Technology in the Synthesis of Human Insulin


complications ensuing from the regular injection of a foreign substance, as well as a
projected decline in the production of animal derived insulin. These factors led researchers
to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E.
coli bacterial cell, to produce insulin that is chemically identical to its naturally produced
counterpart. This has been achieved using Recombinant DNA technology. This method
(see fig. 2) is a more reliable and sustainable method than extracting and purifying the
abattoir by-product.




                         Fig. 2: An overview of the recombination process.



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Recombinant DNA Technology in the Synthesis of Human Insulin


   b) Understanding the genetics involved: -

       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.




                                            Fig. 3

   c) Inside the Double Helix: -

       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).DNA (Deoxyribonucleic Acid), which makes up the chromosome, consists of two
long intertwined helices, constructed from a chain of nucleotides, each composed of a sugar
deoxyribose, a phosphate and nitrogen base. There are four different nitrogen bases,
adenine, thymine, cytosine and guanine. The synthesis of a particular protein such as
insulin is determined by the sequence in which these bases are repeated (see fig. 4).




           Fig. 4: DNA strand with the specific nucleotide sequence for Insulin chain B.



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Recombinant DNA Technology in the Synthesis of Human Insulin


    d) Insulin synthesis from the genetic code: -

        The double strand of the eleventh chromosome of DNA divides in two; exposing
unpaired nitrogen bases which are specific to insulin production (see fig. 5).




Fig. 5: Unravelling strand of the DNA of chromosome 11, with the exposed nucleotides coding for the B
chain of Insulin.



Using one of the exposed DNA strands (see fig.6) as a template, messenger RNA forms in
the process of transcription (see fig. 7).




                         Fig 6: A single strand of DNA coding for Insulin chain B.




                                       Fig. 7: The (m) RNA strand.



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Recombinant DNA Technology in the Synthesis of Human Insulin


       The role of the mRNA strand, on which the nitrogen base thymine is replaced by
uracil, is to carry genetic information, such as that pertaining to insulin, from the nucleus
into the cytoplasm, where it attaches to a ribosome (see fig. 8).




                       Fig. 8: Process of translation at the Ribosome.

       The nitrogen bases on the mRNA are grouped into threes, known as codons.
Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to a specific amino
acid, collectively known as an anti-codon (see fig.9) pair with complementary bases (the
codons) on the mRNA.




                                             Fig. 9


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Recombinant DNA Technology in the Synthesis of Human Insulin


The reading of the mRNA by the tRNA at the ribosome is known as translation. A specific
chain of amino acids is formed by the tRNA following the code determined by the mRNA.
The base sequence of the mRNA has been translated into an amino acid sequence which
link together to form specific proteins such as insulin.

    e) The Vector (Gram negative E. coli): -

       A weakened strain of the common bacterium, Escherrichia coli (E. coli) (see fig.
10), an inhabitant of the human digestive tract, is the 'factory' used in the genetic
engineering of insulin.




                  Fig. 10: The insulin is introduced into an E. coli cell such as this.

When the bacterium reproduces, the insulin gene is replicated along with the plasmid a
circular section of DNA (see fig. 11). E. coli produces enzymes that rapidly degrade
foreign proteins such as insulin.

                                                                    By using mutant strains that lack
                                                           these enzymes, the problem is avoided.
                                                           In E. coli, B-galactosidase is the enzyme
                                                           that controls the transcription of the genes.
                                                           To make the bacteria produce insulin, the
                                                           insulin gene needs to be tied to this
                                                           enzyme.



Fig. 11: Electron micrograph of the Vector's plasmid.


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Recombinant DNA Technology in the Synthesis of Human Insulin


    f) Inside the genetic engineer's toolbox: -

                                                           Restriction      enzymes,     naturally
                                                    produced by bacteria, act like biological
                                                    scalpels (see   fig.12),   only    recognising
                                                    particular stretches of nucleotides, such as
                                                    the one that codes for insulin.




Fig 12: An analogous look at Restriction enzymes.




                                                                     This makes it possible to
                                                             sever certain nitrogen base pairs and
                                                             remove the section of insulin coding
                                                             DNA from one organism's
                                                             chromosome so that it can
                                                             manufacture insulin (See fig. 13).
                                                             DNA ligase is an enzyme which
                                                             serves as a genetic glue, welding the
                                                             sticky ends of exposed nucleotides
                                                             together.




                    Fig: 13




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Recombinant DNA Technology in the Synthesis of Human Insulin


   g) Manufacturing Humulin: -

       The first step is to chemically synthesise the DNA chains that carry the specific
nucleotide sequences characterising the A and B polypeptide chains of insulin (see fig. 14).




                Fig. 14: Human insulin structure. Amino acid RNA to DNA conversion

      The required DNA sequence can be determined because the amino acid
compositions of both chains have been charted. Sixty three nucleotides are required for
synthesising the A chain and ninety for the B chain, 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' (see fig. 15) 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.




                                          Fig. 15


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Recombinant DNA Technology in the Synthesis of Human Insulin


       The recombinant plasmids are then introduced into E. coli 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 (see fig. 16).




                                 Fig. 16: The process of mitosis.

       The protein which is formed consists partly of B-galactosidase, joined to either the
A or B chain of insulin (see fig.17). The A and B chains are then extracted from the B-
galactosidase fragment and purified.




                                          Fig. 17


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Recombinant DNA Technology in the Synthesis of Human Insulin


       The two chains are mixed and reconnected in a reaction that forms the disulfide
cross bridges, resulting in pure Humulin - synthetic human insulin (see fig. 18).




                        Fig. 18: Human insulin molecule




Biological implications of genetically engineered Recombinant human
insulin: -
       Human insulin is the only animal protein to have been made in bacteria in such a
way that its structure is absolutely identical to that of the natural molecule. This reduces the
possibility of complications resulting from antibody production. In chemical and
pharmacological studies, commercially available Recombinant DNA human insulin has
proven indistinguishable from pancreatic human insulin. Initially the major difficulty
encountered was the contamination of the final product by the host cells, increasing the risk
of contamination in the fermentation broth. This danger was eradicated by the introduction
of purification processes. When the final insulin product is subjected to a battery of tests,
including the finest radio-immuno assay techniques, no impurities can be detected. The
entire procedure is now performed using yeast cells as a growth medium, as they secrete an
almost complete human insulin molecule with perfect three dimensional structure. This
minimises the need for complex and costly purification procedures.




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Recombinant DNA Technology in the Synthesis of Human Insulin




Clinical Pharmacology: -
        Adequate insulin dosage permits the diabetic patient to utilize carbohydrates and
fats in a comparatively satisfactory manner. Regardless of concentration, the action of
insulin is basically the same: to enable carbohydrate metabolism to occur and thus to
prevent the production of ketone bodies by the liver. Although, under usual circumstances,
diabetes can be controlled with doses in the vicinity of 40 to 60 units or less, an occasional
patient develops such resistance or becomes so unresponsive to the effect of insulin that
daily doses of several hundred, or even several thousand, units are required. Patients who
require doses in excess of 300 to 500 units daily usually have impaired insulin receptor
function.
        Occasionally, a cause of the insulin resistance can be found (such as
hemochromatosis, cirrhosis of the liver, some complicating disease of the endocrine glands
other than the pancreas, allergy, or infection), but in other cases, no cause of the high
insulin requirement can be determined.
        Humulin R (U-500) is unmodified by any agent that might prolong its action;
however, clinical experience has shown that it frequently has a time action similar to a
repository insulin preparation. It takes effect rapidly but has a relatively long duration of
activity following a single dose (up to 24 hours) as compared with other Regular insulin.
This effect has been credited to the high concentration of the preparation. The time course
of action of any insulin may vary considerably in different individuals or at different times
in the same individual. As with all insulin preparations, the duration of action of Humulin
R (U-500) is dependent on dose, site of injection, blood supply, temperature, and physical
activity.




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Recombinant DNA Technology in the Synthesis of Human Insulin


Advantages of Humulin over Insulin: -


   Earlier insulin required for diabetes was extracted from pancreas of slaughtered cattle,
pigs or salmon. The process was quite tedious and difficult and yields of insulin would be
low. This extracted insulin in some patients, developed allergy or other side effects due to
foreign protein. Due to disadvantages of animal insulin and advantages of humulin,
humulin is regarded superior to animal insulin Humulin is considered better than animal
insulin because:
    Humulin is absorbed more rapidly and show its effectiveness in short duration.
    Humulin causes fewer allergic and autoimmune reactions as compared to animal
       insulin.
    Humulin is less expensive than animal insulin


The issue of hypoglycemic complications in the administration of human
insulin: -
   Hypoglycemia (too little glucose in the blood) is one of the most frequent adverse
events experienced by insulin users. It can be brought about by;
   1. Taking too much insulin
   2. Missing or delaying meals
   3. Exercising or working more than usual
   4. An infection or illness (especially with diarrhea or vomiting)
   5. A change in the body's need for insulin
   6. Diseases of the adrenal, pituitary, or thyroid gland, or progression of kidney or liver
       disease
   7. Interactions with other drugs that lower blood glucose, such as oral hypoglycemics,
       salicylates (for example, aspirin), sulfa antibiotics, and certain antidepressants
   8. Consumption of alcoholic beverages


       Since porcine insulin was phased out, and the majority of insulin dependent patients
are now treated with genetically engineered recombinant human insulin, doctors and


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Recombinant DNA Technology in the Synthesis of Human Insulin


patients have become concerned about the increase in the number of hypoglycemic
episodes experienced. Although hypoglycemia can be expected occasionally with any type
of insulin, some people with diabetes claim that they are less cognisant of attacks of
hypoglycemia since switching from animal derived insulin to Recombinant DNA human
insulin. In a British study, published in the 'Lancet", hypoglycemia was induced in patients
using either pork or human insulin, the researchers found "no significant difference in the
frequency of signs of hypoglycemia between users of the two different types of insulin."
       An anecdotal report from a British patient, who had been insulin dependent for
thirty years, stated that she began experiencing recurring, unheralded hypoglycemia only
after substituting Recombinant DNA human insulin for animal derived insulin. After
switching back to pork insulin to ease her mind, she hadn't experienced any unannounced
hypoglycemia. One organization that was the manufacturer of human insulin, noted that a
third of people with diabetes, who have been insulin dependent for over ten years, "lose
their hypoglycemic warning signals, regardless of the type of insulin they are taking."


Symptoms of mild to moderate hypoglycemia may occur suddenly and can include:-
            sweating
            dizziness
            palpitation
            tremor
            hunger
            restlessness
            tingling in the hands, feet, lips, or tongue
            lightheadedness
            inability to concentrate
            headache
            drowsiness
            sleep disturbances
            anxiety
            blurred vision
            slurred speech
            depressive mood
            irritability


                                                                                          26
Recombinant DNA Technology in the Synthesis of Human Insulin


            abnormal behavior
            unsteady movement
            personality changes
            Signs of severe hypoglycemia can include:
            disorientation
            unconsciousness
            seizures
            death
Therefore, it is important that assistance be obtained immediately


Lipodystrophy: -
       Rarely, administration of insulin subcutaneously can result in lipoatrophy
(depression in the skin) or lipohypertrophy (enlargement or thickening of tissue)... A
change in your injection technique may help alleviate the problem.


Allergy to insulin: -


Local Allergy: - Patients occasionally experience redness, swelling, and itching at the site
of injection of insulin. This condition, called local allergy, usually clears up in a few days
to a few weeks. In some instances, this condition may be related to factors other than
insulin, such as irritants in the skin cleansing agent or poor injection technique.


Systemic Allergy: - Less common, but potentially more serious, is generalized allergy to
insulin, which may cause rash over the whole body, shortness of breath, wheezing,
reduction in blood pressure, fast pulse, or sweating. Severe cases of generalized allergy
may be life threatening.




                                                                                         27
Recombinant DNA Technology in the Synthesis of Human Insulin




Precaution: -
General: - Every patient exhibiting insulin resistance who requires Humulin R (U-500) for
control of diabetes should be under close observation until appropriate dosage is
established. The response will vary among patients. Some patients can be controlled with a
single dose daily; others may require 2 or 3 injections per day. Most patients will show a
"tolerance" to insulin, so that minor variations in dosage can occur without the
development of untoward symptoms of insulin shock.
Insulin resistance is frequently self-limited; after several weeks or months during which
high dosage is required, responsiveness to the pharmacologic effect of insulin may be
regained and dosage can be reduced.
Information for Patients: - Patients should be instructed regarding their dosage and
should be reminded that this formulation requires the administration of a smaller volume of
solution than is the case with less concentrated formulations.
Laboratory Tests: - Blood and urine glucose, glycohemoglobin, and urine ketones should
be monitored frequently.
Drug Interactions: - The concurrent use of oral hypoglycemic agents with Humulin R (U-
500) is not recommended since there are no data to support such use.
Pregnancy-Teratogenic Effects: - No reproduction studies have been conducted in
animals, and there are no adequate and well-controlled studies in pregnant women. It would
be anticipated that the benefits of this insulin preparation would outweigh any risk to the
developing fetus.
Nonteratogenic Effects: - Insulin does not cross the placenta as does glucose.
Labor and Delivery: - Careful monitoring of the patient is required, since the insulin
requirement may decrease following delivery.
Nursing Mothers: - It is not known whether insulin is excreted in significant amounts in
human milk. Because many drugs are excreted in human milk, caution should be exercised
when Humulin R (U-500) insulin injection is administered to a nursing woman.
Pediatric Use: - There are no special precautions relating to the use of this insulin
formulation in the pediatric age group.



                                                                                      28
Recombinant DNA Technology in the Synthesis of Human Insulin




Dosage and administration: -
Humulin R (U-500) should only be administered subcutaneously. It is inadvisable to inject
Humulin R (U-500) intravenously because of possible inadvertent over dosage.
It is recommended that an insulin syringe or tuberculin-type syringe be used for the
measurement of dosage. Variations in dosage are frequently possible in the insulin-resistant
patient, since the individual is unresponsive to the pharmacologic effect of the insulin.
Nevertheless, accuracy of measurement is to be encouraged because of the potential danger
of the preparation.


Storage: -
Insulin should be stored in a refrigerator but not in the freezer. If refrigeration is not
possible, the bottle of insulin that you are currently using can be kept unrefrigerated as long
as it is kept as cool as possible (below 86°F [30°C]) and away from heat and light. Do not
use insulin if it has been frozen. Do not use a bottle of insulin after the expiration date
stamped on the label.


Conclusion: -
Insulin required for diabetes was extracted from pancreas of slaughtered cattle, pigs or
salmon. The process was too long and difficult and its yield would be low.
This extracted insulin in some patients, developed allergy or other side effects due to
foreign protein.
Humulin is absorbed more rapidly and show effectiveness in short duration. Fewer allergic
and autoimmune reactions and less expensive.
Due to disadvantages of animal insulin, and advantages of humulin, it is superior to animal
insulin.




                                                                                          29
Recombinant DNA Technology in the Synthesis of Human Insulin




References: -
1. ADA, Diabetes Information. Retrieved July 9, 2008,
from ADA Website : http://www.diabetes.org/about-diabetes.jsp
2. Hurd, R. (4/26/07). Diabetes Risk Factors. Retrieved July 10, 2008, from University
Of Maryland Medical Center Web site: http://www.umm.edu/ency/article/002072.htm
3. Hirst, Jenny (1997). Adverse effects of human insulin. Retrieved July 17, 2008, from
Health-science-Spirit Web site: http://www.health-science-spirit.com/insulin.html
4. (2003). Carbs Information. Retrieved July 17, 2008, from Insulin: synthetic human
and animal insulin Web site: http://www.carbs-information.com/insulin-synthetic.htm
5. Shump, S (1997). The Johns Hopkins Guide to Diabetes. Baltimore, Maryland: The
Johns Hopkins University Press.
6. Greene, A. (2002, 06, 14). Long term complication. Retrieved July 17, 2008, from Dr.
Greene Caring for the next generation Web site: http://www.drgreene.com/21_1396.html
7. Bloom, A (1982). Diabetes Explained. Lancaster, England: Redwood Burn Limited
8. http://www.littletree.com.au/dna.htm
9. Encyclopedia of Science and Technology (McGraw-Hill).
10. Genetic Engineering, Compton's Interactive Encyclopedia.
11. Galloway, J.A. - Chemistry and Clinical Use of Insulin.
12. Insulin - Grolier Eloctronic Publishing.
13. CSIRO Research of Australia: 8 Biotechnology, pg 63.
14. HMge - Human insulin from second generation genetic engineering.
15. Source of figures is Novo-Nordisk promotional brochure
16. http://www.druglib.com/druginfo/humulin/
17. http://www.xamplified.com/biology/humulin-synthetic-insulin/
18. http://www.wikipedia.com
19. Hellman B, Gylfe E, Grapengiesser E, Dansk H, Salehi A (2007). "[Insulin oscillations-
-clinically important rhythm. Antidiabetics should increase the pulsative component of the
insulin release]" (in Swedish). Lakartidningen 104 (32-33): 2236–9.



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