Phages can be used in two formats of phage DNA and phage display vaccines, with the display technology making the largest contribution to vaccine design and screening large phage libraries.
Phage display: exogenous DNA is inserted into a specific site in the nucleotide sequence encoding one of the phage coat proteins (major, minor). When phage particles infect their bacterial host and express their genes, guest amino acids encoded by foreign DNA are expressed as part of the relevant coat protein. As a consequence, a fusion protein is displayed on the exposed surface of phage virion.
Residues are able to interact with a wide variety of external target molecules, and the phage display tech results from its ability to establish a physical connection between phenotype and genotype. This link makes it possible for researchers to isolate target-avid ligans displayed on the phage.
This technology has the capacity to produce very large libraries of peptides and the tech is already scalable, fast, and reliable. To construct a phage display library, a highly diverse pool of randomized oligonucleotide sequences are splitted into one of the phage coat proteins. This when cloned can create billions of unique displayed peptide ligands where each phage virion in the library displays an individual type of guest peptide on its surface.
The small size of phage virions allows researchers to screen a larger number of different particles in each round of phage display, and one of the most contributing factors to the widespread application of phage display is the convenience for screening target-specific binders.
For specified peptide phage display libraries, randomly fragmented DNA derived from the genetic material of a bacteria is incorporated into the phage genome. This can be applied so that phage particles display on the surface fragments of natural proteins. These are more likely to mount an antibody response but in many cases, the majority of clones are nonfunctional.
Screening phage display libraries for enrichment of peptides that specifically bind to the target of interest is done through an affinity selection-based strategy called biopanning. In biopanning, a population of phage particles is incubated with the target for a specified period of time. The target is then exposed to a series of purification steps where phages whose displayed peptides bind to the target are captured, whereas the unbound or weakly-bound phages are removed.
The benefit of these phage particles is that they are far more stable in harsh conditions, where then target-specific binders are identified through DNA sequencing of the selected phages. During rounds of biopanning, the stringency of selection can be intensified by progressively increasing the number or duration of washes in order to isolate phage clones with the highest affinity towards the target. This can all be automated with robotics through which the peptide library can be incubated using cell fluorescence.
