Human Insulin Gene Expression and Production

 
Cloning and expression of Human
insulin gene
 
Diabetes and the role of insulin
 
Diabetes mellitus
Characterized by the excretion of large amount of
glucose in urine.
Resulted from the body’s inability to make sufficient
insulin.
Insulin
A hormone excreted from the β cells in the pancreas,
and involved in the regulation of blood sugar
At high level of blood glucose the insulin is secreted
into the bloodstream, circulates throughout the body,
and signals the liver and muscle cells to remove the
excess glucose.
 
Diabetes and the role of insulin
 
Insulin recognizes specific insulin receptors on
particular cells and initiates a cascade of reactions
leading to 
glucose uptake
 
In absence of glucose uptake, individual cell will
starve even plenty of glucose is available
 
Starving cells use fats as a primary source of
energy leading to synthesis of ketone bodies
 
The ketone bodies and the glucose (high
concentration) excreted in urine with huge
amount  of water.
 
 
 
Factors led to the development of
human insulin production by bacteria
 
 
The steady increase in the incidence of
diabetes
Allergic reactions due to animal insulin in
some patients
Bacterial cells are easy to grow in mass
quantity to provides a ready source for product
 
The first human gene product manufactured by recombinant DNA
technology was human insulin, called Humulin, which was licensed for
therapeutic use in 1982 by the 
U.S. Food and Drug Administration
(FDA)
, the government agency responsible for regulating the safety of
food and drug products and medical devices.
 
In 1977, scientists at Genentech, the San Francisco biotechnology
company cofounded in 1976 by Herbert Boyer and Robert Swanson
isolated and cloned the gene for insulin and expressed it in bacterial
cells.
 
Genentech, short for “genetic engineering technology,” is also generally
regarded as the world’s first biotechnology company.
 
Steps of insulin production from genetically
engineered bacteria 1.  Obtain the gene for insulin
from human DNA
 
Insert the gene into bacterial cells (restriction
endonuclease, vector, transformation)
 
Select the cells that have the desired gene
 
Induce the bacterial cells to express the inserted
gene to produce insulin
 
Collect and purify the insulin
 
Obtaining the insulin gene
 
Finding the insulin gene in the DNA (finding
needle in haystack
Isolation of mRNA encoding insulin from
pancreatic β cells
mRNA is more abundant in β cells than the
insulin gene
Eukaryotic mRNA contains a poly A tail attached
to one end of each RNA
This poly A tail can be used to isolate the mRNA
from other nucleic acids after breaking the β cell
 
 
 
 
 
Conversion of mRNA to  a complementary
strand cDNA by reverse transcriptase enzyme
 
 
Problem associated with obtaining
the insulin from mRNA
 
It is synthesized by the β cells of the pancreas as
preproinsulin (inactive)
It is stored as proinsulin (inactive) and not
released until it is needed by the body
Preproinsulin includes the signals necessary to
direct the protein to storage. As it is being stored,
the part of the protein responsible for signaling
storage is specifically removed, leading to
formation of proinsulin.
Proinsulin is the form that is stored.
 
 
 
 
 
Active insulin is formed only when the body in
need for insulin then proinsulin is activated by
two specific cuts
The active form of insulin is actually two
separate protein chains (A & B) held together
by bonds
 
Clusters of cells embedded in the pancreas synthesize a
precursor polypeptide known as preproinsulin.
 
As this polypeptide is secreted from the cell, amino acids are
cleaved from the end and the middle of the chain.
These cleavages produce the mature insulin molecule, which
contains two polypeptide chains (the 
A 
and 
B 
chains) joined by
disulfide bonds.
 
The 
A 
subunit contains 21 amino acids, and the 
B 
subunit
contains 30.
 
In the original bioengineering process, synthetic genes that
encode the 
A 
and 
B 
subunits were constructed by
oligonucleotide synthesis (63 nucleotides for the 
A 
polypeptide
and 90 nucleotides for the 
B 
polypeptide).
 
Each synthetic oligonucleotide was inserted into a separate vector,
adjacent to the 
lacZ 
gene encoding the bacterial form of the enzyme b-
galactosidase. When transferred to a bacterial host, the 
lacZ 
gene and
the adjacent synthetic oligonucleotide were transcribed and translated
as a unit.
 
The product is a 
fusion protein
—that is, a hybrid protein consisting
of the amino acid sequence for b-galactosidase attached to the amino
acid sequence for one of the insulin subunits
 
The fusion proteins were purified from bacterial extracts and treated
with cyanogen bromide, that cleaves the fusion protein from the b-
galactosidase.
 
When the fusion products were mixed, the two insulin subunits
spontaneously united, forming an intact, active insulin molecule.
 
Is there a way to get the cells to make active
insulin instead of inactive preproinsulin?
 
If each of two chains of the active insulin could
be made separately  and then properly assembled
with the other, active insulin could be produced
This is the way recombinant insulin was finally
produced.
A gene encoding information for one insulin chain
was cloned into bacterial cells. The chain was
expressed and purified from these  cells
 The information for the other chain was cloned
separately, ultimately  providing purified second
chains
 
 
 
 
 
 
The two strands were mixed outside the cells and
bound to each other in proper fashion to form the
active insulin molecule
 
Thus, cloned insulin was really produced as a mixture
of two  separately cloned products.
 
Because the sequence of amino acids that comprise the
two chains was known, this information was used in
conjunction with the genetic code to work backward i.e.
protein to DNA
 
An advance technique in which fully automated
computer process (DNA synthesizer) is finally made it
possible to complete the cloning of insulin
 
To obtain insulin genes, the nucleotides sequences of
the two chains of the insulin protein are assembled by
using DNA synthesizer rather than the mRNA reverse
transcription.
 
Inserting the insulin gene into bacterial
cells
 
The synthesized DNA segments are incorporated within
vector eg plasmid
The synthesized DNAs do not contain the same
recognition site to make the same sticky ends so small
artificial pieces of DNA (linkers) that contain the same
restriction enzyme recognition sequence as the plasmid
are added
The linkers are attached to the synthesized DNAs by
ligase enzyme then digested with the same restriction
enzymes to give the same sticky ends.
 The vector is then introduced into bacterial cells
(competent) by  transformation
 
 
Inducing the expression of insulin in
bacterial cells.
 
Insulin may be degraded by bacterial cells.
Once produced by bacterial cells, insulin may
be degraded by protease enzyme
Use genetically engineered bacteria deficient
of protease gene.
 
 
Purifying the product
(downstream processing)
 
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The process of cloning and expressing the human insulin gene has revolutionized the production of insulin for treating diabetes. By using genetically engineered bacteria, the human insulin gene is inserted, expressed, and purified to create insulin for therapeutic use. This innovation has overcome challenges such as allergic reactions to animal insulin and increased the availability of insulin for patients with diabetes.


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  1. Cloning and expression of Human Cloning and expression of Human insulin gene insulin gene

  2. Diabetes and the role of insulin Diabetes mellitus Characterized by the excretion of large amount of glucose in urine. Resulted from the body s inability to make sufficient insulin. Insulin A hormone excreted from the cells in the pancreas, and involved in the regulation of blood sugar At high level of blood glucose the insulin is secreted into the bloodstream, circulates throughout the body, and signals the liver and muscle cells to remove the excess glucose.

  3. Diabetes and the role of insulin Insulin recognizes specific insulin receptors on particular cells and initiates a cascade of reactions leading to glucose uptake In absence of glucose uptake, individual cell will starve even plenty of glucose is available Starving cells use fats as a primary source of energy leading to synthesis of ketone bodies The concentration) excreted amount of water. ketone bodies and the glucose with huge (high in urine

  4. Factors led to the development of human insulin production by bacteria The steady increase in the incidence of diabetes Allergic reactions due to animal insulin in some patients Bacterial cells are easy to grow in mass quantity to provides a ready source for product

  5. The first human gene product manufactured by recombinant DNA technology was human insulin, called Humulin, which was licensed for therapeutic use in 1982 by the U.S. Food and Drug Administration (FDA), the government agency responsible for regulating the safety of food and drug products and medical devices. In 1977, scientists at Genentech, the San Francisco biotechnology company cofounded in 1976 by Herbert Boyer and Robert Swanson isolated and cloned the gene for insulin and expressed it in bacterial cells. Genentech, short for genetic engineering technology, is also generally regarded as the world s first biotechnology company.

  6. Steps of insulin production from genetically engineered bacteria 1. Obtain the gene for insulin from human DNA Insert the gene into bacterial cells (restriction endonuclease, vector, transformation) Select the cells that have the desired gene Induce the bacterial cells to express the inserted gene to produce insulin Collect and purify the insulin

  7. Obtaining the insulin gene Finding the insulin gene in the DNA (finding needle in haystack Isolation of mRNA encoding insulin from pancreatic cells mRNA is more abundant in cells than the insulin gene Eukaryotic mRNA contains a poly A tail attached to one end of each RNA This poly A tail can be used to isolate the mRNA from other nucleic acids after breaking the cell

  8. Conversion of mRNA to a complementary strand cDNA by reverse transcriptase enzyme

  9. Problem associated with obtaining the insulin from mRNA It is synthesized by the cells of the pancreas as preproinsulin (inactive) It is stored as proinsulin (inactive) and not released until it is needed by the body Preproinsulin includes the signals necessary to direct the protein to storage. As it is being stored, the part of the protein responsible for signaling storage is specifically removed, leading to formation of proinsulin. Proinsulin is the form that is stored.

  10. Active insulin is formed only when the body in need for insulin then proinsulin is activated by two specific cuts The active form of insulin is actually two separate protein chains (A & B) held together by bonds

  11. Clusters of cells embedded in the pancreas synthesize a precursor polypeptide known as preproinsulin. As this polypeptide is secreted from the cell, amino acids are cleaved from the end and the middle of the chain. These cleavages produce the mature insulin molecule, which contains two polypeptide chains (the A and B chains) joined by disulfide bonds. The A subunit contains 21 amino acids, and the B subunit contains 30. In the original bioengineering process, synthetic genes that encode the A and B subunits were constructed by oligonucleotide synthesis (63 nucleotides for the A polypeptide and 90 nucleotides for the B polypeptide).

  12. Each synthetic oligonucleotide was inserted into a separate vector, adjacent to the lacZ gene encoding the bacterial form of the enzyme b- galactosidase. When transferred to a bacterial host, the lacZ gene and the adjacent synthetic oligonucleotide were transcribed and translated as a unit. The product is a fusion protein that is, a hybrid protein consisting of the amino acid sequence for b-galactosidase attached to the amino acid sequence for one of the insulin subunits The fusion proteins were purified from bacterial extracts and treated with cyanogen bromide, that cleaves the fusion protein from the b- galactosidase. When the fusion products were mixed, the two insulin subunits spontaneously united, forming an intact, active insulin molecule.

  13. Is there a way to get the cells to make active insulin instead of inactive preproinsulin? If each of two chains of the active insulin could be made separately and then properly assembled with the other, active insulin could be produced This is the way recombinant insulin was finally produced. A gene encoding information for one insulin chain was cloned into bacterial cells. The chain was expressed and purified from these cells The information for the other chain was cloned separately, ultimately providing purified second chains

  14. The two strands were mixed outside the cells and bound to each other in proper fashion to form the active insulin molecule Thus, cloned insulin was really produced as a mixture of two separately cloned products.

  15. Because the sequence of amino acids that comprise the two chains was known, this information was used in conjunction with the genetic code to work backward i.e. protein to DNA An advance technique in which fully automated computer process (DNA synthesizer) is finally made it possible to complete the cloning of insulin To obtain insulin genes, the nucleotides sequences of the two chains of the insulin protein are assembled by using DNA synthesizer rather than the mRNA reverse transcription.

  16. Inserting the insulin gene into bacterial cells The synthesized DNA segments are incorporated within vector eg plasmid The synthesized DNAs do not contain the same recognition site to make the same sticky ends so small artificial pieces of DNA (linkers) that contain the same restriction enzyme recognition sequence as the plasmid are added The linkers are attached to the synthesized DNAs by ligase enzyme then digested with the same restriction enzymes to give the same sticky ends. The vector is then introduced into bacterial cells (competent) by transformation

  17. Inducing the expression of insulin in bacterial cells. Insulin may be degraded by bacterial cells. Once produced by bacterial cells, insulin may be degraded by protease enzyme Use genetically engineered bacteria deficient of protease gene.

  18. Purifying the product (downstream processing)

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