Phage display

Phage display is a method for the study of protein-protein, protein-peptide, and protein-DNA interactions that utilizes bacteriophage to connect proteins with the genetic information that encodes them.cite journal |author=Smith GP |title= Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface |journal=Science |volume=228 |issue= 4705 |pages= 1315–1317|year=1985 |doi=10.1126/science.4001944 |pmid=4001944 ] This connection between genotype and phenotype enables large libraries of proteins to 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, [cite journal |author=Smith GP, Petrenko VA |title= Phage display |journal= Chem. Rev. |volume=97 |issue= 2 |pages= 391–410|year=1997 |doi=10.1021/cr960065d] [cite journal |author=Kehoe JW, Kay BK |title= Filamentous phage display in the new millennium |journal= Chem. Rev. |volume=105 |issue= 11 |pages= 4056–4072|year=2005 |doi=10.1021/cr000261r ] though T4, T7, and λ phage have also been used.

Principle

Like the two-hybrid system, phage display is used for the high-throughput screening of protein interactions. 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. Multiple cloning sites are sometimes used to ensure that the fragments are inserted in all three possible frames so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then transformed into male "E. coli" bacterial cells such as TG1 or XL1-Blue "E. coli". The 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. The incorporation of many different DNA fragments into the pIII or pVIII genes generates a library from which members of interest can be isolated.

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. [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. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17088290 PMID: 17088290] ]

General protocol

# Target proteins or DNA sequences are immobilised to the wells of a microtiter plate.
# 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.
# This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
# Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
# 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.
# 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.

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 [ [http://genome.wellcome.ac.uk/doc%5Fwtd020763.html Explanation of "Protein interaction mapping" from The Wellcome Trust] ] . The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)cite journal |author=Hufton SE, Moerkerk PT, Meulemans EV, de Bruïne A, Arends JW, Hoogenboom HR |title=Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands|journal=J. Immunol. Methods |volume=231 |issue=1-2 |pages=39–51 |year=1999 |pmid=10648926 |url=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-418YGFH-5&_user=1543454&_coverDate=12%2F10%2F1999&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053633&_version=1&_urlVersion=0&_userid=1543454&md5=ad85a4991cf0cee17bfec69bc90dd707|doi=10.1016/S0022-1759(99)00139-8] and in searching for protein-DNA interactionscite journal |author=Gommans WM, Haisma HJ, Rots MG |title=Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command|journal=J. Mol. Biol. |volume=354 |issue=3 |pages=507–19 |year=2005 |pmid=16253273 |url=http://www.rug.nl/farmacie/onderzoek/basisEenheden/THErapeuticGeneModulation/publicaties/publicaties2004/2005_6.pdf?as=binary|doi=10.1016/j.jmb.2005.06.082] 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. [cite journal |author=Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U, Strukelj B, Plazar N.|title=Comparison of bacterial and phage display peptide libraries in search of target-binding motif|journal=Appl. Biochem. Biotechnol. |year=2005 |volume=127 |issue=2 |pages=125–31 |doi=10.1385/ABAB:127:2:125] [cite journal |author=Bratkovic T, Lunder M, Popovic T, Kreft S, Turk B, Strukelj B, Urleb U. |title=Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K |journal=Biochem. Biophys. Res. Commun. |year=2005 |volume=332|issue=3 |pages=897–903 |doi=10.1016/j.bbrc.2005.05.028] [cite journal |author=Lunder M, Bratkovic T, Kreft S, Strukelj B |title=Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies |journal=J. Lipid Res. 2005 |volume=46 |issue=7 |pages=1512–6 |doi=10.1194/jlr.M500048-JLR200 |year=2005 |pmid=15863836] . Competing methods for "in vitro" protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.

See also

*Two-hybrid system, an alternative technique for studying protein-protein interactions
*Protein-protein interactions
*mRNA display
*Ribosome display
*Yeast display
*Bacterial display

References

ee also

* [http://dx.doi.org/10.1016/S0076-6879(00)28406-1] 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

* [http://nobelprize.org/nobelfoundation/symposia/chemistry/ncs-2001-2/abstract-smith.html Selection Versus Design in Chemical Engineering]