Dr.

Mohamed El-Azizi
Department of Microbiology and
Biotechnology

Cloning and expression of

insulin gene

Biotechnology: Demystifying the Concepts p (138-

168)
 Compensatory Lectures

Lecture # Original date Compensation Slot/Location

date

Microbiology 7 12/4/2009 5/4/2009 2nd/H5

Microbiology 8 19/4/2009 21/4/2009 5th/H5

Introduction to 4 12/4/2009 14/4/2009 5th/H5

Biotechnology
 Tools of Gene Cloning
4. Detection of the Cloned Gene by Colony
Hybridization Technique
The cloned gene can be detected by a technique known as Colony hybridization:
1- Cells from each colonies are transferred to a filter paper by pressing the paper against the surface of
the agar plate.

2- Treating the paper with denature solution which lyses the cells and releases the DNA.

3- Denatured DNA adheres to the filter.

4- The filter is treated with a labeled probe (specific to the target gene) to allow hybridization between
the probe and the denatured DNA of the gene (if it exists).

5- The filter is washed with fresh solution to remove the excess free probes (not bound specifically to
denatured DNA).

6- Emission of the labeled probe forms black spot on X-ray film.

7. Comparison of the location of the probe signal on the film to the location of the colony on the original
agar plate identifying colony containing DNA complementary to the probe (target gene)
 Cloning DNA sequences that encode
prokaryotic proteins

Gene expression in prokaryotes

All processes take place in the cytoplasm

 Cloning DNA sequences that encode
prokaryotic proteins
Making gene library (bank) in prokaryotes
Subdivision of the genomic DNA into clonable elements and inserting
them into host cells is called gene library (clone bank, gene bank).
1. The complete DNA of an organism is cut with restriction
endonucleases to fragments.
2. Each fragment is inserted into a vector (one may contain the target
gene)
3. Inserting the vectors with DNA segments into host cells

4. After a library is created, the clone (s) with target sequence is

identified.
 Cloning DNA sequences that encode
Eukaryotic proteins

Gene expression in Eukaryotes

In the nucleus

In the cytoplasm
Cloning DNA sequences that encode
Eukaryotic proteins
 Cloning DNA sequences that encode
Eukaryotic proteins
Making cDNA library in eukaryotes
1. Isolation of mRNA encoding the protein.

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

enzyme.

3. The mRNA stands are then chemically degraded leaving single stranded cDNA.

4. The cDNA is converted to double stranded DNA by enzyme DNA polymerase.

5. The resultant DNA contains the sequences of the genes from which mRNAs were
transcribed are cut with restriction endonucleases, inserted into vectors, and finally
into host cells

6. After a library is created, the clone (s) with target sequence is identified.
T F A target gene from prokaryotic cell can be obtained by
making gene library.

T F In cloning of an eukaryotic gene, the target gene

can be obtained directly from the eukaryotic
chromosome and inserted into E. coli.
 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.
 Diabetes and the role of insulin

- Non insulin-dependent diabetes is treated by diet and weight

reduction.
- Insulin-dependent diabetes is treated by injection of controlled
amount of insulin.
- Until recently, insulin was obtained by extraction from animals
pancreases (cow, pig), obtained from slaughterhouses.
 Cloning and expression of insulin gene

Factors led to the production of human insulin in

bacteria.
1. The steady increase in the incidence of diabetes.
2. The reduction of red meat consumption leads to
decrease in animal pancreases available.
3. Allergic reactions due to animal insulin in some
patients.
4. Bacterial cells are easy to grow in mass quantity to
provides a ready source for product.
 Cloning and expression of insulin gene
Steps of insulin production from genetically engineered bacteria
1. Obtain the gene for insulin from human DNA.
2. Insert the gene into bacterial cells (restriction endonuclease, vector,
transformation)
3. Select the cells that have the desired gene.
4. Induce the bacterial cells to express the inserted gene to produce
insulin.
5. Collect and purify the insulin.
 Cloning and expression of insulin gene
1- 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 mRNA.
- This poly A tail can be used to isolate the mRNA from
other nucleic acids after breaking the β cell.
 Cloning and expression of insulin gene
1- Obtaining the insulin gene
- Conversion of mRNA to a complementary strand cDNA
by reverse transcriptase enzyme.
- The mRNA stands are then chemically degraded leaving
single stranded cDNA.
- The cDNA is converted to double stranded DNA by
enzyme DNA polymerase.
- The resultant DNA contains the sequences of the genes
from which mRNAs were transcribed.
- The more the mRNAs to start with the more cDNA for
insulin will be made.
 Cloning and expression of insulin gene

Problem associated with obtaining the insulin from mRNA.

A. Eukaryotic cells often modify proteins after they are synthesized
for activation
- It may involves addition of lipid or carbohydrate groups to the
protein (glycosylation)
- It may involves removal of portion (s) from the polypeptide chain.
- Unmodified proteins are non functional and prokaryotes can not
perform these modifications.
 Cloning and expression of insulin gene

A. Problem associated with obtaining the insulin from

mRNA.
Regarding insulin
a. It is synthesized by the β cells of the pancreas as
preproinsulin (inactive).
b. 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.
 Cloning and expression of insulin gene

A. Problem associated with obtaining the insulin from

mRNA.
Regarding insulin
- 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.
 Cloning and expression of insulin gene
What does this have to do with our cloned insulin?
- It is the preproinsulin form that is encoded by insulin mRNA.
- Thus, if the gene for insulin, which we obtained from mRNA, is
cloned into bacterial cells and expressed as protein, the bacterial cells
will produce the inactive preproinsulin, rather than active insulin.
 Cloning and expression of insulin gene

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
made.
- 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.
 Cloning and expression of insulin gene

Is there a way to get the cells to make active insulin instead of

inactive preproinsulin?
- 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.
 Cloning and expression of insulin gene

How was the genetic information for each individual chain

obtained?
- There is only a single gene for preproinsulin, the protein that gets
processed into active insulin.
- 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.
 Cloning and expression of insulin gene

2- Inserting the insulin gene into bacterial cells

- The synthesized DNA segments are incorporated within vector e.g.
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.
 Cloning and expression of insulin gene

3- Selecting cells with the desired gene.

Distinguish bacteria that have taken plasmid (with or without the
DNA insert).
- First, we distinguish bacteria that have taken up any plasmid DNA
regardless it is the construct or not by growing these bacteria on agar
containing ampicillin.

- Bacteria that grow on ampicillin are those that have taken plasmids.
 Cloning and expression of insulin gene

3- Selecting cells with the desired gene.

Distinguish bacteria that have taken up plasmids with an insert and those that
have taken plasmids with no insert
- By using the blue-white screening ( β galactosidase and X-gal).
- Competent bacteria are allowed to grow on agar containing ampicillin and X-gal.
- The plasmid contains the gene encodes for β galactosidase enzyme which act on
X-gal (white color) and gives blue compound.
- The plasmid contains the restriction enzyme recognition site in the β galactosidase
gene sequence.
- Recircled plasmids taken by bacteria contain intact β galactosidase. So X-gal will
be cleaved to blue compound leading to formation of blue bacterial colonies.
- Plasmids with insert (insulin gene) taken by bacteria contain disrupted β
galactosidase. So X-gal will be intact leading to formation of white bacterial
colonies (target).
Cloning and expression of insulin gene
Cloning and expression of insulin gene
 Cloning and expression of insulin gene
4- Inducing the expression of insulin in
bacterial cells.
Initiating transcription of the
inserted insulin gene
- Transcription initiation in
prokaryotes requires a promotor
- Can be done by inserting the
insulin gene into the restriction
sites which is adjacent to a
prokaryotic promotor.
 Cloning and expression of insulin gene
4- 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.

Insulin may be toxic to bacterial cells

- Try other bacteria.
 Cloning and expression of insulin gene

5- Purifying the product (downstream processing)

- In order to separate two molecules, we need to exploit
differences between them with respect to their properties. For
example,
Size: One might be small, while the other is large.
Charges: One might have a positive charge, and one might have a
negative charge.
Solubility: One might dissolve readily in water, while another might
not.
Size exclusion chromatography (also known as gel filtration
chromatography) to separate two proteins of different sizes.