Methods and Concepts in the Life Sciences/Phage Display
Phage display[edit | edit source]
Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to display the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.
The most common bacteriophages used in phage display are M13 and fd filamentous phage, though T4, T7, and λ phage have also been used.
In the case of M13 filamentous phage display, the DNA encoding the protein or peptide of interest is ligated into the pIII or pVIII gene, encoding either the minor or major coat protein, respectively. Multiple cloning sites are sometimes used to ensure that the fragments are inserted in all three possible reading frames so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then inserted into E. coli. If a phagemid vector is used (a simplified display construct vector) phage particles will not be released from the E. coli cells until they are infected with helper phage, which enables packaging of the phage DNA and assembly of the mature 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 eluted, used to produce more phage (by bacterial infection with helper phage) and so produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as 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 sequenced to identify the relevant, interacting proteins or protein fragments.
