· or even mutated proteins. E.coli is used

·        
Method when we can use an organism to
produce a large sufficient amount of target protein for several purposes (such
as scientific studying or therapeutic).

·        
Protein expression can be done with: bacterial
expression system, yeast expression system, mammalian expression system and
insect expression system.

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·        
Several
protein are created via POE and are might use for large scale application
such as:

ü  Hormonal production (insulin).  

ü  Enzymes production.

ü  Tissue and bone growth factors

ü  Clotting factors production.

ü  Production of recombinant protein
vaccines

ü  Monoclonal, diagnostic, therapeutic
antibodies.

ü  Interferon production.

ü  Studying of a mutant protein

ü  Biotechnology, medicine and
research.

·        
Its advantages for science:  Allow
generation of large amount of the target protein so availability to study low quantity,
rare, toxic or even mutated proteins. 

 

E.coli is used as host to replicate cDNA in order to
generate large population of cell that contains identical DNA.

·        
First, the target DNA
must be cut it with restriction enzyme as well as the plasmid vector and then must
be ligated with DNA ligaseà (cDNA is formed)

·        
Second, the E.coli is treated
either by calcium chloride or heat shock to allow pick up of plasmid and
genetically transformed.

·        
Third, all the E.coli
cells are placed on a grow media that contain antibiotics.

·        
Fourth, only bacteria
with plasmid can survive, each one reproduces to make a colony.

·        
Fifth, a colony grows up
to make many more identical bacteria so finally many more identical copies of
the target DNA.

 

 

 

 

 

A.     The pET system is characterized by:

·        
Producing
high quantity of the desired protein (the desired protein can comprise more
than 50 % of the total cell protein a few hours after induction).

·        
Ability
to regulate the transcription of the target gene (make target gene silent in
the un-induced state)

 

B.      The pEt system mechanism:

 First
of all, the pET system composed of modified strain of E.coli with pET vector
and inducer called IPTG.

·        
The
pET vector contains the following:

ü 
T7 promoter à this come from
bacteriophage

ü  LacZ
gene à help to know whether or not our gene is
been sub-cloned in our vector “give blue color if the gene is not inserted”

ü  Multiple
cloning sites à contain several restriction enzyme site
to allow our DNA to be inserted.

ü  F1
origin of replication à to create single-stranded DNA.

ü  Antibiotic resistance
gene (ampicillin) à to
select the bacteria that only our plasmid.

ü 
Origin of
replication.

In the pET vector, Lac repressor binds to the lac operator (Lac
O) and prevents the promoter from the expression of the target gene, but in the
presence of inducer (IPTG) the Lac repressor will not be binding with lac operator
(Lac O) so the promoter will be activated and the target gene will be
expressed.  

In the E.coli
cell, the genome of the cell is modified by
inserting gene for T7 RNA polymerase (gene 1) and this gene expression is also
controlled by the activation of the promoter after adding an inducer (IPTG).

Then we are going to transform the
vector into this modified type of E.coli cell, and the following will happened:

1.      First, Adding the inducer (IPTG) to
the strain of E.coli so that will cause the Lac repressor to not be binding
with the Lac operator so promoter will be activated and the T7 RNA polymerase
gene (gene 1) is transcribed so RNA polymerase is expressed.   

2.      Second, The RNA polymerase will
recognize and bind to the T7 promoter in the pET vector so this will cause the
production of mRNA and the over-expression of our target protein.

 

C.      Purpose of adding IPTG: the gene 1 must be expressed to produce RNA
polymerase to express our target gene. These two genes expression are
controlled by promoters which is inactivated due to Lac repressor and Lac
operator binding. However, the only way to dissociate them and activate the
promoter is by adding IPTG in the culture to serves as inducer for the system.