Iycee Charles de Gaulle Summary Peptide expressed on the surface of filamentous

Peptide expressed on the surface of filamentous

Peptide phage display a molecular
technique that allows peptides and proteins to be expressed on the surface of
filamentous bacteriophages by inserting the gene for a specific protein on the
surface coat of a given bacteriophage. In order for the peptide to be displayed
on the surface of the phage, the gene fragment must be fused with the coat
protein of the bacteriophage. This technique was developed in 1985 by George P.
Smith when a specific peptide was fused to the gene III of a phage. This will
allow for a connection between genotype and phenotype, the genotype being the
DNA and the phenotype being the peptide being displayed. There
has also been the construction of phage display libraries which can be used
with randomly selected peptides to help identify and analyze those peptides
that are able to bind to specific substrates. These peptide- phage display
libraries can be used in future applications such as antibody epitope mapping,
selection of various molecular targets, such as specific organs, and highly
specific drug production and delivery.   

Biopanning is an application of peptide phage
display which is a high affinity selection technique that will select
peptides with the ability to bind to a specific or given target.
Biopanning is performed by incubating the phage display library with a very
specific target/ligand with the goal of being able to identify the proteins or
peptides that are able to successfully bind to that target. After being
incubated for an extended amount of time, an elution must be performed to
separate any unbound phages and to isolate those phages that successfully bound
to the target; this washing process is performed with either acidic or highly
basic solutions.  In order to obtain those phages with a
particularly high affinity for the target, the entire process of biopanning is
repeated about three to five times. Once these phages have
been selected, the coding for the displayed peptide can be analyzed, which will
allow for identification of that peptide.Filamentous bacteriophages are used as a vector in the process
of peptide phage display because of the characteristics of these phages. The
filamentous bacteriophages, including those of the Ff species (f1, M13 and fd),
employed in phage display most commonly infect Escherichia coli. These filamentous bacteriophages contain
single stranded DNA and have a pilus that allows the bacteriophage to attach
itself to E. coli and to allow for further infection. After infecting a given
host in nature, theses bacteriophages will undergo the lytic life cycle. In order for propagation of the virus, the virion must be
able to exit the host cell as well as phages will keep the host cell intact and
alive. The phage will use the
machinery of the host cell for self-replication and, when replication is
complete, the newly made phages will slowly bud out of the cell membrane. This
slow release of phages allows for the survival of the host cell, which is
necessary for the virion to replicate, making this an ideal life cycle for the
process of biopanning.  Another bacteriophage that can be used in this
process is bacteriophage T7 which consists of double- stranded DNA. Bacteriophage
M13 is most commonly used in phage display, but T7 is more stable in some
potentially unfavorable conditions, such has high temperature or extreme pH levels.

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There are many possible medical
applications that phage display could positively influence. For instance, in
vivo biopanning is the process of identifying and isolating phage- peptide
libraries that have are able to bind with specific targets in a given animal. The
peptide- phage display must be circulated in the blood of the animal for an
extended amount to allow for identification of the target tissue as well as the
binding to said tissue. After circulation, tissue samples are extracted from
the animal and analyzed for potentially bound phages displaying peptides. More
research and development of this process could lead to more specific drug
targeting as well as less systemic absorption.

Phage display has been used in the
detection of the intestinal infection, cholera. This infection is cause by the
bacterium Vibrio cholerae and, when untreated, can cause severe
dehydration, shock and death to its victims. The current methods and technology
of detecting an infection are too time consuming and there is need for a
quicker and less expensive method, which led to the use of peptide phage
display. V. cholera produces an endotoxin, cholera toxin (CTX), which is the
major cause of the symptoms brought on by the infection. The endotoxin is
comprised of two subunits, CTX A and CTX B. CTX A subunit is responsible for
the bacteria’s toxicity while CTX B is responsible for binding to the receptors
of the intestinal cells. The B subunit is non-toxic and is the target of
current identifying and monitoring methods of the infectious disease.            This
experiment questioned whether phage display would quickly identify the presence
of the CTX B subunit of a cholera infection. Phage M13 was used in this
experiment and underwent three rounds of biopanning with the unique peptides
that are able to recognize the CTX B subunit. After the three rounds of
biopanning, it was found that there was not one dominant peptide that was
responsible for binding but six phages that were selected for further
identification with specific amino acid sequences. ELISA was performed on these
six phages in order to further investigate the binding properties to CTX B. The
results of ELISA showed that there was binding affinity to this subunit, and
with an increase in the concentration of the CTX B subunit there was also an
increase in binding affinity. Overall, this experiment was
successful in using peptide phage display to bind and identify a cholera
infection. This data can now be applied to enhance the current methods of
detecting and monitoring a cholera infection.

Another experiment using peptide
phage display was conducted with the question if detection of high arsenic
As(III) levels in drinking water was possible. Arsenic pollution is found in
underdeveloped countries where accurate and inexpensive methods of detection
are required but are not feasible for these countries. This experiment
questioned whether peptide phage display would identify the presence of arsenic
in the water in a way that is cost effective and is not effected on the
presence of any other metals.             Several
rounds of biopanning were performed with a negative and positive control.
Current methods and techniques that identify high levels of arsenic can be
easily effected by the presence of other toxic and nontoxic metals. To assure
specific binding to arsenic, screening against metal cations, such as cadmium,
ferric and cupric oxide, was performed as a negative control. This negative
screening would assure that the selected phage library used would
preferentially bind to As (III) over these foreign cations. Four rounds of
biopanning were performed with the negative controls and the resulting unbound
phages to these foreign cations were collected and used in the next round of
screening. After the negative screenings, the unbound phages were then
incubated with the target metal, As(III). The phages with the highest affinity
for arsenic were identified after three rounds of biopanning. Resulting were
twenty randomly selected phages with As(III)- binding peptide and of those
twenty, twelve were successfully sequenced in five monoclonal groups. Of those
twelve phages, eight of them exhibited the same peptide sequence. This shared
peptide sequence could indicate a higher As(III) affinity. In conclusion, this
experiment provided data showing that, with the use of peptide phage display,
detection of high levels of As(III) is possible.

With the previous two experiments,
both successful, there is great potential in the applications of phage display.
Peptide phage display has many advantages over current methods being used to
isolate peptides/proteins of harmful molecules or infections. Peptide- phage
display is less expensive than various methods with a higher accuracy rate. In
order for peptide phage display to be successful, the molecules must bind with
high affinity, which means the phage library is highly specific and will only
bind with the given target. A disadvantage of peptide- phage display would be
several rounds of biopanning that must be undergone in order to obtain such
high affinity. After biopanning, high affinity binding may not even be
obtained. If this binding does not occur, a new phage library must be obtained
and the biopanning process must be started from the beginning. If successful,
peptide-phage display could greatly influence the ways medicine is developed
and administered.             Phage
display has had a positive impact on the detection, isolation and analysis of
the peptides that have successfully bound to a given target. The further
investigation of peptide- phage display could radically change the way diseases
or infections are treated, as well as further analysis of peptides or proteins.
There is an increased need for phage display, whether it be with peptides,
antibodies or enzymes. The combination of phage display with these three molecules
can be used in oncologic studies, the detection, monitoring and treatment of
diseases, as well as the detection of harmful chemicals that may be present in
drinking water.