Recombinant
DNA Technology
in the Synthesis
of Human Insulin
The nature and
purpose of synthesising human
insulin.
Since
Banting and Best discovered the
hormone, insulin in 1921.(1)
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,(2)
regulates the use and storage of
food, particularly
carbohydrates.
Fig. 1 Fluctuations in diabetic
person's blood glucose levels,
compared with healthy
individuals. Source: Hillson,R.
- Diabetes: A beyond basics
guide, pg.16.
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 complications ensuing
from the regular injection of a
foreign substance,(3) as
well as a projected decline in
the production of animal derived
insulin.(4)
These factors led researchers to
consider synthesising Humulin
by inserting the insulin
gene into a suitable vector, the
E. coli bacterial cell, to
produce an 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(5)
method than extracting and
purifying the abattoir
by-product.
Fig. 2 An overview of the
recombination process. Source:
Novo - Nordisk promotional
brochure,pg 6.
Understanding the
genetics involved.
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.(6)
Fig. 3 Source: Chance, R. and Frank
B. - Research, development,
production and safety of
Biosynthetic Human Insulin.
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
Deoxyribolnucleic 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.(7) 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. Source: Based on the
diagram in Watson, J.D., Gilman,
M., Witkovski, J., Zoller, M. -
Recombinant DNA, pg 22.
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. Source:
Based on the diagram in Watson,
J.D., Gilman, M., Witkovski, J.,
Zoller, M. - Recombinant DNA, pg
22.
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.
Source: Novo-Nordisk promotional
brochure, pg 13.
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. Source:
Novo-Nordisk promotional
brochure, pg 13.
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.
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.
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. Source:
Novo-Nordisk promotional
brochure, pg 16 .
When the
bacterium reproduces, the
insulin gene is replicated along
with the plasmid,(8) 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.(9)
Fig. 11 Electron micrograph of the
Vector's plasmid. Source:
Watson, J.D., Gilman, M.,
Witkovski, J., Zoller, M. -
Recombinant DNA, pg 73.
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.
Inside
the genetic engineer's toolbox.
Restriction
enzymes, naturally produced by
bacteria, act like biological
scalpels(10) (see
fig.12), only recognising
particular stretches of
nucleotides, such as the one
that codes for insulin.(11)
Fig 12 An analogous look at
Restriction enzymes. Source:
CSIRO Research of Australia No.
8.
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 Source: Watson, J.D.,
Gilman, M., Witkovski., Zoller,
M. - Recombinant DNA, pg 78.
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. Source: Genetic
Engineering Activities, pg 176.
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.
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.
Source: Novo-Nordisk promotional
brochure, pg 11.
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 Source: Watson, J.D.,
Gilman, M., Witkovski, J.,
Zoller, M. - Recombinant DNA, pg
456.
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.
Source: Source: Watson, J.D.,
Gilman, M., Witkovski, J.,
Zoller, M. - Recombinant DNA, pg
456.
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.(12)
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,(13)
no impurities can be detected.(14) 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.
The issue of
hypoglycaemic complications in
the administration of human
insulin.
Since
porcine insulin was phased out,
and the majority of insulin
dependent patients are now
treated with genetically
engineered recombinant human
insulin, doctors and patients
have become concerned about the
increase in the number of
hypoglycaemic episodes
experienced.(15)
Although hypoglycaemia (
hypoglycemia , insulin shock,
insulin reaction ) can be
expected occasionally with any
type of insulin, some people
with diabetes claim that they
are less cognisant of attacks of
hypoglycaemia ( hypoglycemia ,
insulin shock, insulin reaction
) since switching from animal
derived insulin to Recombinant
DNA human insulin.(16) In
a British study, published in
the 'Lancet", hypoglycaemia
( hypoglycemia , insulin shock,
insulin reaction ) was induced
in patients using either pork or
human insulin, The researchers
found "no significant
difference in the frequency of
signs of hypoglycaemia (
hypoglycemia , insulin shock,
insulin reaction ) between users
of the two different types of
insulin."(17)
An
anecdotal report from a British
patient who had been insulin
dependent for thirty years,
stated that she began
experiencing recurring,
unheralded hypoglycaemia (
hypoglycemia , insulin shock,
insulin reaction ) 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 hypoglycaemia (
hypoglycemia , insulin shock,
insulin reaction ). Eli Lilly
and Co., a manufacturer of human
insulin, noted that a third of
people with diabetes, who have
been insulin dependent for over
ten years, "lose their
hypoglycaemic warning signals,
regardless of the type of
insulin they are taking."(18)
Dr Simon
P. Wolff of the University
College of London said in an
issue of Nature ,
"As far as I can make out,
there's no fault (with the human
insulin)." He
concluded, "I do
think we need to have a study to
examine the possible risk."(19)
Although
the production of human insulin
is unarguable welcomed by the
majority of insulin dependent
patients, the existence of a
minority of diabetics who are
unhappy with the product cannot
be ignored. Although not a new
drug, the insulin derived from
this new method of production
must continue to be studied and
evaluated, to ensure that all
its users have the opportunity
to enjoy a complication free
existence.
Endnotes:
1.
Banting - Grolier Electronic
Publishing.
2. Encyclopedia of Science and
Technology (McGraw-Hill).
3. ibid.
4. Galloway, J.A. - Chemistry
and Clinical Use of Insulin.
5. op. cit.
6. Insulin - Grolier Eloctronic
Publishing.
7. Genetic Engineering,
Compton's Interactive
Encyclopedia.
8. op. cit.
9. ibid.
10. CSIRO Research of Australia:
8 Biotechnology, pg 63.
11. ibid.
12. Galloway, J.A. - Chemistry
and Clinical Use of Insulin, pg
106
13. HMge - Human insulin from
second generation genetic
engineering.
14. ibid.
15. Price, J. - Lawsuit over
human insulin looming in
Britain, 26-4-92.
16. ibid.
17. ibid.
18. ibid.
19. ibid.
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