Phage Display is a powerful method for finding interesting peptide interactions through affinity screening of a large random peptide library.

Bacterial viruses (phages) have been genetically modified to express a variety of peptides which then are allowed to interact with a target of interest. One of the advantages of using phages is that they can be amplified in bacteria. This allows us to repeat the experiment with narrower pools of peptides and thus finding more specific bindings. Phage genomes are small and known which allows easy determination of the final specific peptide by DNA sequencing of the viruses.

Since the method of phage display was developed in 1985 [1] it has been applied for various purposes such as studying antibodies, observing immune system responses, and evolving antibodies for therapy purposes. It has also been used for whole cell recognition (often used for targeting cancer) and in-vivo screening of individual tissues for endothelial cell markers [2] More commonly, phage display is performed with a protein as the target, in order to find a ligand that would affect the function of the protein.

For the purpose of our project we need a way to detect the parasites. A peptide binding specifically to the exterior of the strongyle would fulfill this purpose. Since we are working with an under-researched organism, starting with a random peptide library seemed reasonable. The only similar organisms that have been used in this method were C. elegens, but as it is a free living nematode whereas strongyles are endoparasites we had reason to believe that the exterior of the membranes would differ greatly due to the different environments the different species inhabit.

Using a random phage library streamlined our work. Phages ensure a link between the DNA sequence and the physical peptides. Furthermore the indigenous behavior of lysogenic phages allowed visualizing and following the procedure as well as enabling amplification of the peptides upon infecting bacteria. Thus, in spite of lack of a premade protocol we decided to try and apply phage display for characterizing our nematodes.

Experiment

New Application

Unknown genome/proteome

Due to our target strongyle having an unknown genome/proteome, selection of a single target protein is not feasible. Efforts could have been made to separate surface proteins from the organism, but such an approach does not provide any guarantees of the extracted proteins displaying correct conformations. To preserve each of the possible protein targets in a physiological state, the entire organism was used for the experiment.

Accessible high throughput screening

Having decided to use a whole organism as the target, the next challenge is finding a way to deal with the plethora of exposed surface proteins. Attempting to identify possible ligands one at a time would have been prohibitively laborious, and a method had to be selected that could screen binding compounds in a massively parallel manner. Several such methods exist, but most would require specialized equipment and/or comprehensive precursor libraries. Phage display is a simple yet robust approach to the same problem.

Immobilization hard for whole worms

To be able to wash away unbound phages, while retaining the bound phages, existing protocols employ various methods for immobilizing the target. Since the target of our experiment is the surface of an entire organism rather than a single protein, the common methods of fixing the target were ineffective, and a new method had to be developed. The implemented solution relies on filter inserts for eppendorf tubes. The filters simultaneously act as a substitute for well plates and an immobilization method.

Experiment procedure

With the experiment necessitating the use of phage display on an entire organism, no existing protocol was entirely applicable. This meant that we had to adapt a protocol as much as it was possible and introduce completely new solutions to problems unique to our application. We based our adapted protocol on the product manual included in the Ph.D.™-12 Phage Display Peptide Library Kit ordered from New Englands BioLabs.

For a detailed description of our protocol click here. Our whole organism phage display experiments started with preparation of the containers that the whole procedure were performed in microcentrifuge tubes with filter inserts. The preparation consisted of blocking the tubes with blocking buffer to prevent non-specific interactions.
To avoid selecting for phage with affinity for the plastic and not the organism, an affinity screening for the tubes was performed before introducing the target. After collecting the phage elute that doesn’t bind to the tubes, we performed our first affinity screening against the small strongyle. This was done by introducing the 12-mer peptides expressing phages to the strongyle placed in the filter tubes. Unbound phages were then washed away followed by elution and collection of the bound phages with an acidic buffer. All washing and elution steps were performed in the filter tubes, where liquid during centrifugation could pass through the filters, leaving the strongyle still on the top of the filter.

In the next step of the experiment, phage titering was carried out, to visualise the amount of infectious phages present in the elute. The elute phage with affinity to the strongyle were then amplified in the E.coli ER2738 host strain, by adding the phage to the strain in early log-phase.
Phage and bacterias were then separated with the help of centrifugation to discard E. coli and the phage were extracted by precipitation.
The success of the amplification was examined by a new round of phage titering. Once aware of the phage titer, the next round of this previously described procedure consisting of affinity screening to the strongyle, washing and elution, phage titering, amplification of phage and a new round of phage titering was performed.

After the third round of affinity screening, single plaques, (consisting of single phage clones) were picked from the titering plates and amplified separate. The phages DNA from the amplified plaques were then extracted and purified.

Sample titers from Series 1

Figure 4: Titered Eluate: Panning 1.

Figure 5: Titered Eluate: Panning 2

Figure 6: Titered Eluate: Panning 3.

Unfortunately the ELISA results were inconclusive due to noticable chromogenic signaling in the filter tubes without any contact to either phages nor strongyles. Since the antibodies used were M13 monoclonal the possibility of the them interacting with the filter or plastic is highly unlikely. One factor that may have contributed to the false ELISA signal could be the prolonged exposure to the blocking buffer in the filter-tubes. Due to its alkaline nature and the stability of the filter being between pH 4-8, the prolonged exposure may have caused filter degradation. The degradation may have also prevented the antibodies to be successfully washed away thus exhibiting the false signal.

Figure 7: Negative control of phage ELISA, containing ONLY monoclonal M13 antibodies and NO phage.

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