Phage display

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Phage display is a test used to screen for protein-protein interactions and protein-DNA interactions[1] by integrating many genetic sequences from a DNA library into the genome of a suitable phage.

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[edit] Principle

Like the two-hybrid system, phage display is used for the high-throughput screening of protein interactions. The principle of this method is based on the ligation of a target DNA fragment into the vector form of the pIII or pVIII gene of a filamentous phage. This DNA fragment is then expressed on the surface of the phage particle as part of its minor (pIII) or major (pVIII) coat protein. If several DNA fragments are incorporated into the pIII or pVIII, then this becomes a library of those DNA fragments. The phage gene and insert DNA hybrid is then transformed into male E. coli bacterial cells such as TG1 E. coli cells. The phage particles will not be released upon maturation of the TG1 E. coli cells until they are infected with helper phage with a multiplicity of infection of 5 (i.e. each bacterial cell is infected with no more than 5 helper phage particles) to extrude the DNA-containing phage which will in turn display the relevant protein fragment as part of their outer coat. Via multiple cloning sites, the fragments are inserted in all three possible frames to ensure that the cDNA fragment is translated in the proper frame.

By immobilising a relevant DNA or protein target(s) to the surface of a 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.

Recent work published by Chasteen et al., shows that use of the helper phage can be eliminated by using a novel 'bacterial packaging cell line' technology.[2]

[edit] General protocol

  1. Target proteins or DNA sequences are immobilised to the wells of a microtiter plate.
  2. Many genetic sequences are expressed in a bacteriophage library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage.
  3. This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
  4. Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
  5. Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant (i.e. non-binding phage) than were present in the initial mixture.
  6. The DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.

[edit] Applications

The applications of this technology include determination of interaction partners of a protein (which would be used as the immobilised phage "bait" with a DNA library consisting of all coding sequences of a cell, tissue or organism) so that new functions or mechanisms of function of that protein may be inferred[3]. The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)[4] and in searching for protein-DNA interactions[1] using specially-constructed DNA libraries with randomised segments.

Phage display is also a widely used method for in vitro protein evolution (also called protein engineering). As such, phage display is a useful tool in drug discovery. It is used for finding new ligands (enzyme inhibitors, receptor agonists and antagonists) to target proteins. [5] [6] [7]. Competing methods for in vitro protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.

[edit] See also

[edit] References

  1. ^ a b Gommans WM, Haisma HJ, Rots MG (2005). "Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command". J. Mol. Biol. 354 (3): 507–19. doi:10.1016/j.jmb.2005.06.082. PMID 16253273. 
  2. ^ Chasteen L, Ayriss J, Pavlik P, Bradbury AR. Eliminating helper phage from phage display. Nucleic Acids Res. 2006;34(21):e145. Epub 2006 Nov 6.PMID: 17088290
  3. ^ Explanation of "Protein interaction mapping" from The Wellcome Trust
  4. ^ Hufton SE, Moerkerk PT, Meulemans EV, de Bruïne A, Arends JW, Hoogenboom HR (1999). "Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands". J. Immunol. Methods 231 (1-2): 39–51. doi:10.1016/S0022-1759(99)00139-8. PMID 10648926. 
  5. ^ Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U, Strukelj B, Plazar N.Comparison of bacterial and phage display peptide libraries in search of target-binding motif.Appl Biochem Biotechnol. 2005 Nov;127(2):125-31.
  6. ^ Bratkovic T, Lunder M, Popovic T, Kreft S, Turk B, Strukelj B, Urleb U.Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K.Biochem Biophys Res Commun. 2005 Jul 8;332(3):897-903.
  7. ^ Lunder M, Bratkovic T, Kreft S, Strukelj B.Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies.J Lipid Res. 2005 Jul;46(7):1512-6.

[edit] See also

  • [1] Phage display for selection of novel binding peptides, Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000), Methods Enzymol., 328, 333–363

Chasteen L, Ayriss J, Pavlik P, Bradbury AR. Eliminating helper phage from phage display. Nucleic Acids Res. 2006;34(21):e145. Epub 2006 Nov 6.