Peptide expressed on the surface of filamentous

Peptide phage display a moleculartechnique that allows peptides and proteins to be expressed on the surface offilamentous bacteriophages by inserting the gene for a specific protein on thesurface coat of a given bacteriophage. In order for the peptide to be displayedon the surface of the phage, the gene fragment must be fused with the coatprotein 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 willallow for a connection between genotype and phenotype, the genotype being theDNA and the phenotype being the peptide being displayed. Therehas also been the construction of phage display libraries which can be usedwith randomly selected peptides to help identify and analyze those peptidesthat are able to bind to specific substrates.

These peptide- phage displaylibraries can be used in future applications such as antibody epitope mapping,selection of various molecular targets, such as specific organs, and highlyspecific drug production and delivery.   Biopanning is an application of peptide phagedisplay which is a high affinity selection technique that will selectpeptides with the ability to bind to a specific or given target.Biopanning is performed by incubating the phage display library with a veryspecific target/ligand with the goal of being able to identify the proteins orpeptides that are able to successfully bind to that target. After beingincubated for an extended amount of time, an elution must be performed toseparate any unbound phages and to isolate those phages that successfully boundto the target; this washing process is performed with either acidic or highlybasic solutions.  In order to obtain those phages with aparticularly high affinity for the target, the entire process of biopanning isrepeated about three to five times.

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Once these phages havebeen selected, the coding for the displayed peptide can be analyzed, which willallow for identification of that peptide.Filamentous bacteriophages are used as a vector in the processof peptide phage display because of the characteristics of these phages. Thefilamentous bacteriophages, including those of the Ff species (f1, M13 and fd),employed in phage display most commonly infect Escherichia coli. These filamentous bacteriophages containsingle stranded DNA and have a pilus that allows the bacteriophage to attachitself to E. coli and to allow for further infection. After infecting a givenhost in nature, theses bacteriophages will undergo the lytic life cycle. In order for propagation of the virus, the virion must beable to exit the host cell as well as phages will keep the host cell intact andalive. The phage will use themachinery of the host cell for self-replication and, when replication iscomplete, the newly made phages will slowly bud out of the cell membrane.

Thisslow release of phages allows for the survival of the host cell, which isnecessary for the virion to replicate, making this an ideal life cycle for theprocess of biopanning.  Another bacteriophage that can be used in thisprocess is bacteriophage T7 which consists of double- stranded DNA. BacteriophageM13 is most commonly used in phage display, but T7 is more stable in somepotentially unfavorable conditions, such has high temperature or extreme pH levels.There are many possible medicalapplications that phage display could positively influence. For instance, invivo biopanning is the process of identifying and isolating phage- peptidelibraries that have are able to bind with specific targets in a given animal. Thepeptide- phage display must be circulated in the blood of the animal for anextended amount to allow for identification of the target tissue as well as thebinding to said tissue. After circulation, tissue samples are extracted fromthe animal and analyzed for potentially bound phages displaying peptides.

Moreresearch and development of this process could lead to more specific drugtargeting as well as less systemic absorption. Phage display has been used in thedetection of the intestinal infection, cholera. This infection is cause by thebacterium Vibrio cholerae and, when untreated, can cause severedehydration, shock and death to its victims. The current methods and technologyof detecting an infection are too time consuming and there is need for aquicker and less expensive method, which led to the use of peptide phagedisplay. V.

cholera produces an endotoxin, cholera toxin (CTX), which is themajor cause of the symptoms brought on by the infection. The endotoxin iscomprised of two subunits, CTX A and CTX B. CTX A subunit is responsible forthe bacteria’s toxicity while CTX B is responsible for binding to the receptorsof the intestinal cells. The B subunit is non-toxic and is the target ofcurrent identifying and monitoring methods of the infectious disease.            Thisexperiment questioned whether phage display would quickly identify the presenceof the CTX B subunit of a cholera infection. Phage M13 was used in thisexperiment and underwent three rounds of biopanning with the unique peptidesthat are able to recognize the CTX B subunit. After the three rounds ofbiopanning, it was found that there was not one dominant peptide that wasresponsible for binding but six phages that were selected for furtheridentification with specific amino acid sequences.

ELISA was performed on thesesix phages in order to further investigate the binding properties to CTX B. Theresults of ELISA showed that there was binding affinity to this subunit, andwith an increase in the concentration of the CTX B subunit there was also anincrease in binding affinity. Overall, this experiment wassuccessful in using peptide phage display to bind and identify a cholerainfection.

This data can now be applied to enhance the current methods ofdetecting and monitoring a cholera infection. Another experiment using peptidephage display was conducted with the question if detection of high arsenicAs(III) levels in drinking water was possible. Arsenic pollution is found inunderdeveloped countries where accurate and inexpensive methods of detectionare required but are not feasible for these countries. This experimentquestioned whether peptide phage display would identify the presence of arsenicin the water in a way that is cost effective and is not effected on thepresence of any other metals.

            Severalrounds of biopanning were performed with a negative and positive control.Current methods and techniques that identify high levels of arsenic can beeasily effected by the presence of other toxic and nontoxic metals. To assurespecific binding to arsenic, screening against metal cations, such as cadmium,ferric and cupric oxide, was performed as a negative control. This negativescreening would assure that the selected phage library used wouldpreferentially bind to As (III) over these foreign cations. Four rounds ofbiopanning were performed with the negative controls and the resulting unboundphages to these foreign cations were collected and used in the next round ofscreening. After the negative screenings, the unbound phages were thenincubated with the target metal, As(III). The phages with the highest affinityfor arsenic were identified after three rounds of biopanning. Resulting weretwenty randomly selected phages with As(III)- binding peptide and of thosetwenty, twelve were successfully sequenced in five monoclonal groups.

Of thosetwelve phages, eight of them exhibited the same peptide sequence. This sharedpeptide sequence could indicate a higher As(III) affinity. In conclusion, thisexperiment 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 toisolate peptides/proteins of harmful molecules or infections. Peptide- phagedisplay is less expensive than various methods with a higher accuracy rate. Inorder for peptide phage display to be successful, the molecules must bind withhigh affinity, which means the phage library is highly specific and will onlybind with the given target. A disadvantage of peptide- phage display would beseveral rounds of biopanning that must be undergone in order to obtain suchhigh affinity. After biopanning, high affinity binding may not even beobtained.

If this binding does not occur, a new phage library must be obtainedand the biopanning process must be started from the beginning. If successful,peptide-phage display could greatly influence the ways medicine is developedand administered.             Phagedisplay has had a positive impact on the detection, isolation and analysis ofthe peptides that have successfully bound to a given target. The furtherinvestigation of peptide- phage display could radically change the way diseasesor 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 moleculescan be used in oncologic studies, the detection, monitoring and treatment ofdiseases, as well as the detection of harmful chemicals that may be present indrinking water.



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