Team:Uppsala/Phage Display
What is phage display?
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 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 repeating the experiment with more narrow pool of peptides thus finding more specific bindings.
The genome of phages is 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 at 1985 [1] it has been applied for various purposes e.g. studying antibodies - response of immune system and evolving antibodies for therapy purposes; whole cell recognition (often used for targeting cancer); in-vivo screening of individual tissues for endothelial cell markers [2]
Most commonly phage display is performed with a protein as a target to find a ligand that would affect the function of the protein.
General Concept
In order to determine a peptide with affinity to a molecule of interest an iterative process of affinity binding and washing, called panning, will yield an end result with a high concentration of high affinity peptides.
Figure 1: Flowchart over the workflow of a typical phage display screening
For the purpose of our project - developing a diagnostic system, a distinct detection of the parasites is a precondition. A peptide binding specifically to the exterior of the strongyle would fulfill this purpose. As we were setting out to detect a under-researched organism, starting with a random peptide library seemed reasonable. Any remote parallels we could have drawn regarding possible surface proteins were with the model organism c.elegans but as it is a free living nematode whereas strongyles are endoparasites we had a reason to believe that the exterior of the membranes would differ greatly due to the different environments the different species are inhabiting.
Approach with viruses serving as vessels for the random peptides streamlined our work. Phages ensured 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
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 insofar as it was possible, and introduce completely new solutions to problems unique to our application.
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 general 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.
Figure 1: Filter tube blocked with Blocking Buffer.
Figure 2: The strongyles are added, exess liquid spun down and discarded.
Figure 3: Phages are added, left to incubate in solution with the worms. The unbound phages are spun down and discarded. The bound phages are subsequently eluted with a acidic buffer
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.
Phage Titering
Phage titering is done after every affinity screening, to assess the amount of phages that bind to the target. By following the titering protocol consisting of plating phages together with mid-log phase bacteria, visible blue plaques are formed on Xgal/IPTGl plates. The goal with plating is to achieve plates with around 100 plaques, which is fulfilled by doing several dilution series of the infected bacteria. The reason for this is that plaques will only increase linearly with added phage, when the multiplicity of infection (MOI) is much less than 1. Also low MOI result in one DNA sequence per plaque. To asses the titre, plaque forming unit (pfu) can be calculate by multiply number of plaques with the bacteria dilution.
Phage titering is also carried out after every phage amplification, now to assess the successfulness of the amplification and to make sure you have a sufficient amount of phage for the subsequente affinity screening.
We did three rounds of panning to select for phages having affinity peptides for the target. The last phage titering could then be used to select single clones to be prepared for sequencing.
Verify affinity and analysis of DNA
So how do we prove that the selected phages clones really binds to our target, the strongyle? With the help of ELISA (enzyme-linked immunosorbent assay) single phage clones affinity to the target could be tested, by screening against plastic binders an assessment regarding which samples are viable can be made. The ELISA was performed in the centrifugal filter-tubes as to expose the phages to the same environment as the panning stage of the process.
After amplification of each chosen plaque, the phage DNA was extracted an analysed before sequencing. We used nanodrop to determine the DNA concentration and gel electrophoresis to determine purity and the size of the DNA.
Results
Three series of panning were performed on Small Strongyles. Each round of panning and amplification were titered and plaque-forming phages were counted to ensure a high enough representation of the library in subsequent pannings.
Sample titers from Series 1
Figure 1: Titered Eluate: Panning 1
Figure 2: Titered Eluate: Panning 2
Figure 3: Titered Eluate: Panning 3
Titering tables
Table 1: Titering results. *Negative panning for interactions with the tube were performed in conjugate with regular panning.
Phage Elisa
The ELISA results even though a chromogenic signal was established in the presence of phages and strongyles the results were inconclusive due to noticing 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 prevented the antibodies to successfully be washed away thus exhibiting the false signal.
Figure 4:Negative control of phage ELISA, containing ONLY monoclonal M13 antibodies and NO phage.
Sequenced Samples and Computational Analysis
Table 2: Samples sent to sequencing. *TUP = Target-Unrelated Peptide.
| Sample | Aligned Sequence | Propagation Advantage | Predicted Polysterene Binder | Known TUP*-Motif | |
|---|---|---|---|---|---|
| Series 1: | EF01122224: TPIFLPTPAQEH | TPIFLPTPAQEH--- | Yes | No | No |
| Series 3: | EF01122218: FSPTQANTIHRW | ---FSPTQANTIHRW | No | No | No |
| EF01122220: VGGTVQSESHRR | --VGGTVQSESHRR- | No | No | No | |
| EF01122222: SMGRTDYVQQLR | -SMGRTDYVQQLR-- | No | No | No | |
| EF01122217: RVQPAHFNVMGQ | --RVQPAHFNVMGQ | No | Yes | No |
Five samples contain pure enough samples of sufficient concentration to satisfy the standard for third party sequencing. Series 1 produced a single samples of high enough quality, Series 2 were all deemed too low for accurate sequencing and Series 3 yieded four samples. The aligment used ClustalW with penalties 25 for gap-creation and 25 for gap-elongation to ensure strict alignments. No clear consensus motifs are distinguishable except slighty hydrophilic residues towards the end of the sequence. Predictive analysis was performed with SAROTUP: Target-Unrelated Peptides Scanners[1].
Conclusions
Due to the fact that our ELISA-test were inconclusive we can not be sure that our peptides are specific binders. Three of the peptides; EF01122218, EF01122220 and EF01122222 shows no predicted or comparative causes for unspecific binding and would be good candidates for future studies.
Links
References
[1]Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228:1315–1317.
[2] Arap, Marco Antonio. (2005). Phage display technology: applications and innovations.Genetics and Molecular Biology, 28(1), 1-9.
[3]. Qiang, Xu, Keyong Sun, Lijun Xing, Yifeng Xu, Hong Wang, Zhengpin Zhou, Juan Zhang, m.fl. ”Discovery of a Polystyrene Binding Peptide Isolated from Phage Display Library and Its Application in Peptide Immobilization”. Scientific Reports 7, nr 1 (december 2017). https://doi.org/10.1038/s41598-017-02891-x.