Editing Phage display (section)

== Principle ==

Like the [[two-hybrid system]], phage display is used for the high-throughput screening of protein interactions. In the case of [[M13 bacteriophage|M13 filamentous phage]] display, the DNA encoding the protein or peptide of interest is [[DNA ligase|ligated]] into the pIII or pVIII gene, encoding either the minor or major [[Protein of the viral capsid|coat protein]], respectively. [[Multiple cloning site]]s are sometimes used to ensure that the fragments are inserted in all three possible [[reading frames]] so that the [[cDNA]] fragment is [[Gene translation|translated]] in the proper frame. The phage gene and insert [[DNA hybridization|DNA hybrid]] is then inserted (a process known as "[[transduction (genetics)|transduction]]") into ''[[Escherichia coli|E. coli]]'' bacterial cells such as TG1, SS320, ER2738, or XL1-Blue ''E. coli''. If a "[[phagemid]]" [[Vector (molecular biology)|vector]] is used (a simplified display construct vector) [[virus|phage particles]] will not be released from the ''E. coli'' cells until they are infected with [[Helper virus|helper phage]], which enables packaging of the phage DNA and assembly of the mature [[virus|virions]] with the relevant protein fragment as part of their outer coat on either the minor (pIII) or major (pVIII) coat protein.
By immobilizing a relevant DNA or protein target(s) to the surface of a [[microtiter plate]] well, a phage that displays a protein that binds to one of those targets on its surface will remain while others are removed by washing. Those that remain can be [[elution|eluted]], used to produce more phage (by [[bacteria]]l infection with helper phage) and to produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as [[Biopanning|'panning']], in reference to the enrichment of a sample of gold by removing undesirable materials.
Phage eluted in the final step can be used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant DNA sequence excised and [[DNA sequencing|sequenced]] to identify the relevant, interacting proteins or protein fragments.{{cn|date=October 2022}}

The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.<ref name="pmid17088290">{{cite journal |vauthors=Chasteen L, Ayriss J, Pavlik P, Bradbury AR | title = Eliminating helper phage from phage display | journal = Nucleic Acids Res. | volume = 34 | issue = 21 | pages = e145 | year = 2006 | pmid = 17088290 | pmc = 1693883 | doi = 10.1093/nar/gkl772 }}</ref>

Elution can be done combining low-pH elution [[buffering agent|buffer]] with sonification, which, in addition to loosening the peptide-target interaction, also serves to detach the target molecule from the immobilization surface. This [[ultrasound]]-based method enables single-step selection of a high-affinity peptide.<ref name="pmid18533899">{{cite journal | vauthors = Lunder M, Bratkovic T, Urleb U, Kreft S, Strukelj B | title = Ultrasound in phage display: a new approach to nonspecific elution | journal = BioTechniques | volume = 44 | issue = 7 | pages = 893–900 | date = June 2008 | pmid = 18533899 | doi = 10.2144/000112759  | doi-access = free }}</ref>