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'''Phage display''' is a laboratory technique for the study of [[Protein–protein interaction|protein–protein]], protein–[[peptide]], and protein–[[DNA]] interactions that uses [[bacteriophage]]s to connect proteins with the [[genetic information]] that [[code|encodes]] them.<ref name="smith">{{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 }}</ref> Phage display was originally described by George P. Smith in 1985, when he demonstrated the display of peptides on [[filamentous phage]] by fusing the peptide of interest on to gene III of filamentous phage.<ref name="smith" /> This technology was further developed and improved by groups at the [[MRC Laboratory of Molecular Biology]] with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins such as [[antibodies]] for [[therapeutic]] [[protein engineering]]. The connection between [[genotype]] and [[phenotype]] enables large [[protein fragment library|libraries of proteins]] to be screened and [[amplification (molecular biology)|amplified]] in a process called ''[[in vitro]]'' selection, which is analogous to [[natural selection]]. The most common bacteriophages used in phage display are [[M13 bacteriophage|M13]] and fd [[filamentous phage]],<ref>{{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 |pmid=11848876}}</ref><ref>{{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 |pmid=16277371 }}</ref> though [[Enterobacteria phage T4|T4]],<ref name=" doi: 10.1016/S0022-2836(02)00298-X">{{cite journal | author = Malys N, Chang DY, Baumann RG, Xie D, Black LW | title = A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction |journal = J Mol Biol |volume = 319 | issue = 2 | pages = 289–304 | year = 2002 | doi =10.1016/S0022-2836(02)00298-X | pmid = 12051907 }}</ref> [[T7 phage|T7]], and [[Lambda phage|λ]] phage have also been used.
'''Phage display''' is a laboratory technique for the study of [[Protein–protein interaction|protein–protein]], protein–[[peptide]], and protein–[[DNA]] interactions that uses [[bacteriophage]]s to connect proteins with the [[genetic information]] that [[code|encodes]] them.<ref name="">{{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= |year=1985 |doi=10.1126/science.4001944 }}</ref> Phage display was originally described by George P. Smith in 1985, when he demonstrated the display of peptides on [[filamentous phage]] by fusing the peptide of interest on to gene III of filamentous phage.<ref name="" /> This technology was further developed and improved by groups at the [[MRC Laboratory of Molecular Biology]] with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins such as [[antibodies]] for [[therapeutic]] [[protein engineering]]. The connection between [[genotype]] and [[phenotype]] enables large [[protein fragment library|libraries of proteins]] to be screened and [[amplification (molecular biology)|amplified]] in a process called ''[[in vitro]]'' selection, which is analogous to [[natural selection]]. The most common bacteriophages used in phage display are [[M13 bacteriophage|M13]] and fd [[filamentous phage]],<ref>{{cite journal |author=Smith GP, Petrenko VA |title= Phage |journal= Chem. Rev. |volume=97 |issue= 2 |pages= 391–410|year=1997 |doi=10.1021/cr960065d }}</ref><ref>{{cite journal |author=Kehoe JW, Kay BK |title= Filamentous phage display in the new millennium |journal= Chem. Rev. |volume=105 |issue= 11 |pages= |year=2005 |doi=10.1021/cr000261r }}</ref> though [[Enterobacteria phage T4|T4]],<ref name="">{{cite journal | author = Malys N, Chang DY, Baumann RG, Xie D, Black LW | title = A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction |journal = J Mol Biol |volume = 319 | issue = 2 | pages = 289–304 | year = 2002 | doi =10.1016/S0022-2836(02)00298-X | pmid = 12051907 }}</ref> [[T7 phage|T7]], and [[Lambda phage|λ]] phage have also been used.


==Principle==
==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 [[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 "[[transformation (genetics)|transformation]]") 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.
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 [[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 "[[transformation (genetics)|transformation]]") 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.
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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.
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.


The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.<ref>{{cite journal | last1 = Chasteen | first1 = L | last2 = Ayriss | first2 = J | last3 = Pavlik | first3 = P | last4 = Bradbury | first4 = AR | title = Eliminating helper phage from phage display. | pmid = 17088290 | author-separator =, | pmc = 1693883 | journal = Nucleic Acids Research | author-name-separator= | doi=10.1093/nar/gkl772 | volume=34 | issue=21 | year=2006 | pages=e145}}</ref>
The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.<ref>{{cite journal | = Chasteen | = | = | = | = | = | pmid = 17088290 | pmc = 1693883 | = 10.1093/nar/gkl772 }}</ref>


==General protocol==
==General protocol==
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==Applications==
==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 region|coding sequences]] of a cell, tissue or organism) so that new functions or mechanisms of function of that protein may be inferred.<ref>[http://genome.wellcome.ac.uk/doc%5Fwtd020763.html Explanation of "Protein interaction mapping" from The Wellcome Trust]</ref> The technique is also used to determine [[tumour]] [[antigens]] (for use in diagnosis and therapeutic targeting)<ref name="hufton">{{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}}</ref> and in searching for [[protein-DNA interaction]]s<ref name="gommans">{{cite 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}}</ref> using specially-constructed DNA libraries with randomised segments.
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 region|coding sequences]] of a cell, tissue or organism) so that new functions or mechanisms of function of that protein may be inferred.<ref>[http://genome.wellcome.ac.uk/doc%5Fwtd020763.html Explanation of "Protein interaction mapping" from The Wellcome Trust]</ref> The technique is also used to determine [[tumour]] [[antigens]] (for use in diagnosis and therapeutic targeting)<ref name="">{{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 |doi=10.1016/S0022-1759(99)00139-8}}</ref> and in searching for [[protein-DNA interaction]]s<ref name="">{{cite 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 |doi=10.1016/j.jmb.2005.06.082}}</ref> 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 [[ligand]]s (enzyme inhibitors, receptor agonists and antagonists) to target proteins.<ref>{{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. | volume=127 |issue=2 |pages=125–31 |doi=10.1385/ABAB:127:2:125 }}</ref><ref>{{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. | volume=332|issue=3 |pages=897–903 |doi=10.1016/j.bbrc.2005.05.028 }}</ref><ref>{{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. |volume=46 |issue=7 |pages=1512–6 |doi=10.1194/jlr.M500048-JLR200 }}</ref>

The invention of antibody phage display by laboratories at the [[MRC Laboratory of Molecular Biology]] led by Greg Winter and [[John McCafferty]] and at The Scripps Research Institute led by Richard Lerner and Carlos F. Barbas revolutionised antibody drug discovery.<ref name="pmid2247164">{{cite journal | author = McCafferty J, Griffiths AD, Winter G, Chiswell DJ | title = Phage antibodies: filamentous phage displaying antibody variable domains | journal = Nature | volume = 348 | issue = 6301 | pages = 552–4 | year = 1990 | month = December | pmid = 2247164 | doi = 10.1038/348552a0 | bibcode = 1990Natur.348..552M}}</ref><ref name="isbn0-87969-740-7">{{cite book | author = Scott JS, Barbas CF III, Burton, DA | title = Phage Display: A Laboratory Manual | publisher = Cold Spring Harbor Laboratory Press | location = Plainview, N.Y | year = 2001 | pages = | isbn = 0-87969-740-7 }}</ref> In 1991, The Scripps group reported the first display and selection of human antibodies on phage.<ref name="pmid1896445">{{cite journal | author = Barbas CF, Kang AS, Lerner RA, Benkovic SJ | title = Assembly of combinatorial antibody libraries on phage surfaces: the gene III site | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 88 | issue = 18 | pages = 7978–82 | year = 1991 | month = September | pmid = 1896445 | pmc = 52428 | doi = 10.1073/pnas.88.18.7978 }}</ref> This initial study described the rapid isolation of human antibody Fab fragements that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy.<ref name="pmid1719545">{{cite journal | author = Burton DR, Barbas CF, Persson MA, Koenig S, Chanock RM, Lerner RA | title = A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 88 | issue = 22 | pages = 10134–7 | year = 1991 | month = November | pmid = 1719545 | pmc = 52882 | doi = 10.1073/pnas.88.22.10134 }}</ref><ref name="pmid1384050">{{cite journal | author = Barbas CF, Björling E, Chiodi F, Dunlop N, Cababa D, Jones TM, Zebedee SL, Persson MA, Nara PL, Norrby E | title = Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 89 | issue = 19 | pages = 9339–43 | year = 1992 | month = October | pmid = 1384050 | pmc = 50122 | doi = 10.1073/pnas.89.19.9339 }}</ref><ref name="pmid7973652">{{cite journal | author = Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, Sawyer LS, Hendry RM, Dunlop N, Nara PL | title = Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody | journal = Science | volume = 266 | issue = 5187 | pages = 1024–7 | year = 1994 | month = November | pmid = 7973652 | doi = 10.1126/science.7973652 }}</ref><ref name="pmid7490758">{{cite journal | author = Yang WP, Green K, Pinz-Sweeney S, Briones AT, Burton DR, Barbas CF | title = CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range | journal = J. Mol. Biol. | volume = 254 | issue = 3 | pages = 392–403 | year = 1995 | month = December | pmid = 7490758 | doi = 10.1006/jmbi.1995.0626 }}</ref><ref name="pmid8170992">{{cite journal | author = Barbas CF, Hu D, Dunlop N, Sawyer L, Cababa D, Hendry RM, Nara PL, Burton DR | title = In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 91 | issue = 9 | pages = 3809–13 | year = 1994 | month = April | pmid = 8170992 | pmc = 43671 | doi = }}</ref>


Following the pioneering disclosures of these laboratories phage display of antibody libraries became a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create the first synthetic human antibody libraries, thereby allowing human antibodies to be created in vitro from synthetic diversity elements.<ref name="pmid1584777">{{cite journal | author = Barbas CF, Bain JD, Hoekstra DM, Lerner RA | title = Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 89 | issue = 10 | pages = 4457–61 | year = 1992 | month = May | pmid = 1584777 | pmc = 49101 | doi = }}</ref><ref name="pmid7694276">{{cite journal | author = Barbas CF, Languino LR, Smith JW | title = High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 90 | issue = 21 | pages = 10003–7 | year = 1993 | month = November | pmid = 7694276 | pmc = 47701 | doi = 10.1073/pnas.90.21.10003 }}</ref>
⚫
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 [[ligand]]s (enzyme inhibitors, receptor agonists and antagonists) to target proteins.<ref>{{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 |pmid=16258189}}</ref><ref>{{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 |pmid=15913550}}</ref><ref>{{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}}</ref>
<ref name=Barbas_1995>{{cite journal | author = Barbas CF, Wagner J | title = Synthetic Human Antibodies: Selecting and Evolving Functional Proteins | journal = Methods | year = 1995 | month = October | volume = 8 | issue = 2 | pages = 94–103|doi=10.1006/meth.1995.9997}}</ref><ref name="pmid7585190">{{cite journal | author = Barbas CF | title = Synthetic human antibodies | journal = Nat. Med. | volume = 1 | issue = 8 | pages = 837–9 | year = 1995 | month = August | pmid = 7585190 | doi = 10.1038/nm0895-837 }}</ref>


Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA ([[adalimumab]]), discovered by [[Cambridge Antibody Technology]] as D2E7 and developed and marketed by [[Abbott Laboratories]]. HUMIRA, an antibody to [[TNF alpha]], was the world's first fully human antibody,<ref name="pmid17420735">{{cite journal | author = Lawrence S | title = Billion dollar babies--biotech drugs as blockbusters | journal = Nat. Biotechnol. | volume = 25 | issue = 4 | pages = 380–2 | year = 2007 | month = April | pmid = 17420735 | doi = 10.1038/nbt0407-380 }}</ref> which achieved annual sales exceeding $1bn.<ref>[http://telegraph.uk-wire.com/cgi-bin/articles/200601251501444434X.html Cambridge Antibody: Sales update | Company Announcements | Telegraph<!-- Bot generated title -->]</ref>
The invention of antibody phage display by laboratories at the [[MRC Laboratory of Molecular Biology]] led by Greg Winter and [[John McCafferty]] and at The Scripps Research Institute led by Richard Lerner and Carlos F. Barbas revolutionised antibody drug discovery.<ref>{{cite journal |author=McCafferty J, Griffiths A.D, Winter G, Chiswell D.J |title=Phage antibodies: filamentous phage displaying antibody variable domains |journal=Nature |issue=63017 |pages=552–554 |doi=10.1038/348552a0 |pmid=2247164 |year=1990 |volume=348 |bibcode=1990Natur.348..552M}}</ref><ref>“Phage Display: A Laboratory Manual” C.F. Barbas, III, D.R. Burton, J.K. Scott, G.J. Silverman, Eds. [[Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York]]; '''2001'''; 736 pages.</ref> In 1991, The Scripps group reported the first display and selection of human antibodies on phage.<ref>“Assembly of combinatorial antibody libraries on phage surfaces: The gene III site” C.F. Barbas III, A.S. Kang, R.A. Lerner, and S.J. Benkovic[[Proc. Natl. Acad. Sci. USA]]; '''1991'''; 88(18); 7978-7982. {{DOI|10.1073/pnas.88.18.7978}}</ref> This initial study described the rapid isolation of human antibody Fab fragements that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy.<ref>“A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals” D.R. Burton, C.F. Barbas III, M.A.A. Persson, S. Koenig, R.M. Chanock, and R.A. Lerner [[Proc. Natl. Acad. Sci. USA]]; '''1991'''; 88(22); 10134-10137. {{DOI|10.1073/pnas.88.22.10134}}</ref><ref>“Recombinant Human Fab fragments neutralize human type 1 immunodeficiency virus in vitro” C.F. Barbas III, E. Bjorling, F. Chiodi, N. Dunlop, D. Cababa, T.M. Jones, S.L. Zebedee, M.A.A. Persson, P.L. Nara, E. Norrby, and D.R. Burton [[Proc. Natl. Acad. Sci. USA]]; '''1992'''; 89(19); 9339-9343. {{DOI| 10.1073/pnas.89.19.9339}}</ref><ref>“Efficient Neutralization of Primary Isolates of HIV-1 by a Recombinant Human Monoclonal Antibody” D.R. Burton, J. Pyati, R. Koduri, S.J. Sharp, G.B. Thornton, P.W.H.I. Parren, L.S.W. Sawyer, M.R. Hendry, N. Dunlop, P.L. Nara, M. Lamacchia, E. Garratty, E.R. Stiehm, Y.J. Bryson, Y. Cao, J.P. Moore, D.D. Ho, and C.F. Barbas III [[Science]]; '''1994'''; 266(5187); 1024-1026. {{DOI| 10.1126/science.7973652}}</ref><ref>“CDR Walking Mutagenesis for the Affinity Maturation of a Potent Human anti-HIV-1 Antibody into the Picomolar RangeZ” W.-P. Yang, K. Green, S. Pinz-Sweeney, A.T. Briones, D.R. Burton, and C.F. Barbas III [[J. Mol. Biol.]]; '''1995'''; 254; 392-403. {{DOI|10.1006/jmbi.1995.0626}}</ref><ref>“In vitro evolution of a neutralizing human antibody to HIV-I to enhance affinity and broaden strain cross-reactivity” C.F. Barbas III, D. Hu, N. Dunlop, L. Sawyer, D. Cababa, R.M. Hendry, P.L. Nara, and D.R. Burton [[Proc. Natl. Acad. Sci. USA]]; '''1994''' 91; 3809-3813.</ref> Following the pioneering disclosures of these laboratories phage display of antibody libraries became a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create the first synthetic human antibody libraries, thereby allowing human antibodies to be created in vitro from synthetic diversity elements.<ref>“Semi-synthetic combinatorial antibody libraries: A chemical solution to the diversity problem” C.F. Barbas III, J.D. Bain, D.M. Hoekstra, and R.A. Lerner [[Proc. Natl. Acad. Sci. USA]]; '''1992'''; 89(10); 4457-4461.</ref><ref>“High Affinity Self-Reactive Human Antibodies by Design and Selection: Targeting the Integrin Ligand Binding Site” C.F. Barbas III, L.R. Languino, and J.W. Smith [[Proc. Natl. Acad. Sci. USA]]; '''1993'''; 90(21); 10003-10007. {{DOI| 10.1073/pnas.90.21.10003}}</ref><ref>“Synthetic Human Antibodies: Selecting and Evolving Functional Proteins” C.F. Barbas III and J. Wagner [[Methods, A Companion to Methods in Enzymology]]; '''1995'''; 8(2); 94-103. {{DOI|10.1006/meth.1995.9997}}</ref><ref>“Synthetic Human Antibodies” C.F. Barbas III [[Nature Medicine]]; '''1995'''; 1; 837-839. {{DOI|10.1038/nm0895-837}}</ref> Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA ([[adalimumab]]), discovered by [[Cambridge Antibody Technology]] as D2E7 and developed and marketed by [[Abbott Laboratories]]. HUMIRA, an antibody to [[TNF alpha]], was the world's first fully human antibody,<ref>[http://www.nature.com/nbt/journal/v25/n4/full/nbt0407-380.html Access : Billion dollar babies|[mdash&#93;|biotech drugs as blockbusters : Nature Biotechnology<!-- Bot generated title -->]</ref> which achieved annual sales exceeding $1bn.<ref>[http://telegraph.uk-wire.com/cgi-bin/articles/200601251501444434X.html Cambridge Antibody: Sales update | Company Announcements | Telegraph<!-- Bot generated title -->]</ref>


Competing methods for ''in vitro'' protein evolution are [[yeast display]], [[bacterial display]], [[ribosome display]], and [[mRNA display]].
Competing methods for ''in vitro'' protein evolution are [[yeast display]], [[bacterial display]], [[ribosome display]], and [[mRNA display]].


== Bioinformatics Resources and Tools ==
== Bioinformatics Resources and Tools ==
Databases and computational tools for [[mimotope]]s have been an important part of phage display study.<ref>{{cite journal|last=Huang|first=J|coauthors=Ru, B, Dai, P|title=Bioinformatics resources and tools for phage display.|journal=Molecules (Basel, Switzerland)|date=2011-01-18|volume=16|issue=1|pages=694–709|pmid=21245805|doi=10.3390/molecules16010694}}</ref> Databases,<ref>{{cite journal|last=Huang|first=J|coauthors=Ru, B, Zhu, P, Nie, F, Yang, J, Wang, X, Dai, P, Lin, H, Guo, FB, Rao, N|title=[[MimoDB]] 2.0: a mimotope database and beyond.|journal=Nucleic Acids Research|date=2011-11-03|pmid=22053087|doi=10.1093/nar/gkr922|volume=40|issue=1|pages=D271–7}}</ref> programs and web servers have been widely used to exclude target-unrelated peptides,<ref>{{cite journal|last=Huang|first=J|coauthors=Ru, B, Li, S, Lin, H, Guo, FB|title=SAROTUP: scanner and reporter of target-unrelated peptides.|journal=Journal of biomedicine & biotechnology|year=2010|volume=2010|pages=101932|pmid=20339521|doi=10.1155/2010/101932|pmc=2842971}}</ref> characterize small molecules-protein interactions and map protein-protein interactions.
Databases and computational tools for [[mimotope]]s have been an important part of phage display study.<ref>{{cite journal|=HuangJ, B, Dai P|title=Bioinformatics resources and tools for phage display|journal= |volume=16|issue=1|pages=694–709|pmid=21245805|doi=10.3390/molecules16010694}}</ref> Databases,<ref>{{cite journal|=HuangJ, B, Zhu P, Nie F, Yang J, Wang X, Dai P, Lin H, Guo FB, Rao N|title=MimoDB 2.0: a mimotope database and beyond|journal=Nucleic Acids |=|pmid=22053087|doi=10.1093/nar/gkr922}}</ref> programs and web servers have been widely used to exclude target-unrelated peptides,<ref>{{cite journal|=HuangJ, B, Li S, Lin H, Guo FB|title=SAROTUP: scanner and reporter of target-unrelated peptides|journal= |=2010|=|pages=101932|pmid=20339521|doi=10.1155/2010/101932}}</ref> characterize small molecules-protein interactions and map protein-protein interactions.


== See also ==
== See also ==
Line 40: Line 44:


==Further reading==
==Further reading==
⚫
*[http://nobelprize.org/nobelfoundation/symposia/chemistry/ncs-2001-2/abstract-smith.html Selection Versus Design in Chemical Engineering]{{dead link|date=July 2010}}
* [https://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&catid=4&sobi2Id=331&Itemid=81 The ETH-2 human antibody phage library]
* [https://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&catid=4&sobi2Id=331&Itemid=81 The ETH-2 human antibody phage library]
*{{cite journal|doi=10.1016/S0076-6879(00)28406-1|title=Phage display for selection of novel binding peptides|last1=Sidhu|first1=S. S.|last2=Lowman|pmid=11075354|first2=H. B.|last3=Cunningham|first3=B. C.|last4=Wells|first4=J. A.|year=2000|journal=Methods Enzymol|volume=328|pages=333–363}}
*{{cite journal|=|title=Phage display for selection of novel binding peptides|=. .|= |= |= |year=2000|= |=}}
⚫
*[http://nobelprize.org/nobelfoundation/symposia/chemistry/ncs-2001-2/abstract-smith.html Selection Versus Design in Chemical Engineering]{{dead link|date=July 2010}}
*{{cite journal|doi=10.1038/348552a0|last1=McCafferty|first1=J.|last2=Griffiths|first2=A.D.|pmid=2247164|last3=Winter|first3=G.|last4=Chiswell|first4=D.J.|year=1990|title=Phage antibodies: filamentous phage displaying antibody variable domains|journal=Nature|volume=348|issue=6301|pages=552−554|bibcode=1990Natur.348..552M}}


{{Protein methods}}
{{Protein methods}}

Revision as of 05:42, 13 May 2012

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.[1] Phage display was originally described by George P. Smith in 1985, when he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene III of filamentous phage.[1] This technology was further developed and improved by groups at the MRC Laboratory of Molecular Biology with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins such as antibodies for therapeutic protein engineering. The 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,[2][3] though T4,[4] 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, 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 (a process known as "transformation") into Escherichia coli (E. coli) bacterial cells such as TG1, SS320, ER2738, or XL1-Blue 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.

The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.[5]

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 phage (i.e. non-binding) 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.

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.[6] The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)[7] and in searching for protein-DNA interactions[8] 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.[9][10][11]

The invention of antibody phage display by laboratories at the MRC Laboratory of Molecular Biology led by Greg Winter and John McCafferty and at The Scripps Research Institute led by Richard Lerner and Carlos F. Barbas revolutionised antibody drug discovery.[12][13] In 1991, The Scripps group reported the first display and selection of human antibodies on phage.[14] This initial study described the rapid isolation of human antibody Fab fragements that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy.[15][16][17][18][19]

Following the pioneering disclosures of these laboratories phage display of antibody libraries became a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create the first synthetic human antibody libraries, thereby allowing human antibodies to be created in vitro from synthetic diversity elements.[20][21] [22][23]

Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA (adalimumab), discovered by Cambridge Antibody Technology as D2E7 and developed and marketed by Abbott Laboratories. HUMIRA, an antibody to TNF alpha, was the world's first fully human antibody,[24] which achieved annual sales exceeding $1bn.[25]

Competing methods for in vitro protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.

Bioinformatics Resources and Tools

Databases and computational tools for mimotopes have been an important part of phage display study.[26] Databases,[27] programs and web servers have been widely used to exclude target-unrelated peptides,[28] characterize small molecules-protein interactions and map protein-protein interactions.

See also

References

  1. ^ a b Smith GP (1985). "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface". Science. 228 (4705): 1315–7. doi:10.1126/science.4001944. PMID 4001944. {{cite journal}}: Unknown parameter |month= ignored (help)
  2. ^ Smith GP, Petrenko VA (1997). "Phage Display". Chem. Rev. 97 (2): 391–410. doi:10.1021/cr960065d. PMID 11848876. {{cite journal}}: Unknown parameter |month= ignored (help)
  3. ^ Kehoe JW, Kay BK (2005). "Filamentous phage display in the new millennium". Chem. Rev. 105 (11): 4056–72. doi:10.1021/cr000261r. PMID 16277371. {{cite journal}}: Unknown parameter |month= ignored (help)
  4. ^ Malys N, Chang DY, Baumann RG, Xie D, Black LW (2002). "A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction". J Mol Biol. 319 (2): 289–304. doi:10.1016/S0022-2836(02)00298-X. PMID 12051907.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ^ Chasteen L, Ayriss J, Pavlik P, Bradbury AR (2006). "Eliminating helper phage from phage display". Nucleic Acids Res. 34 (21): e145. doi:10.1093/nar/gkl772. PMC 1693883. PMID 17088290.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ^ Explanation of "Protein interaction mapping" from The Wellcome Trust
  7. ^ 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. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  8. ^ 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. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  9. ^ Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U, Strukelj B, Plazar N (2005). "Comparison of bacterial and phage display peptide libraries in search of target-binding motif". Appl. Biochem. Biotechnol. 127 (2): 125–31. doi:10.1385/ABAB:127:2:125. PMID 16258189. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  10. ^ Bratkovic T, Lunder M, Popovic T, Kreft S, Turk B, Strukelj B, Urleb U (2005). "Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K". Biochem. Biophys. Res. Commun. 332 (3): 897–903. doi:10.1016/j.bbrc.2005.05.028. PMID 15913550. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  11. ^ Lunder M, Bratkovic T, Kreft S, Strukelj B (2005). "Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies". J. Lipid Res. 46 (7): 1512–6. doi:10.1194/jlr.M500048-JLR200. PMID 15863836. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  12. ^ McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature. 348 (6301): 552–4. Bibcode:1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  13. ^ Scott JS, Barbas CF III, Burton, DA (2001). Phage Display: A Laboratory Manual. Plainview, N.Y: Cold Spring Harbor Laboratory Press. ISBN 0-87969-740-7.{{cite book}}: CS1 maint: multiple names: authors list (link)
  14. ^ Barbas CF, Kang AS, Lerner RA, Benkovic SJ (1991). "Assembly of combinatorial antibody libraries on phage surfaces: the gene III site". Proc. Natl. Acad. Sci. U.S.A. 88 (18): 7978–82. doi:10.1073/pnas.88.18.7978. PMC 52428. PMID 1896445. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  15. ^ Burton DR, Barbas CF, Persson MA, Koenig S, Chanock RM, Lerner RA (1991). "A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals". Proc. Natl. Acad. Sci. U.S.A. 88 (22): 10134–7. doi:10.1073/pnas.88.22.10134. PMC 52882. PMID 1719545. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  16. ^ Barbas CF, Björling E, Chiodi F, Dunlop N, Cababa D, Jones TM, Zebedee SL, Persson MA, Nara PL, Norrby E (1992). "Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro". Proc. Natl. Acad. Sci. U.S.A. 89 (19): 9339–43. doi:10.1073/pnas.89.19.9339. PMC 50122. PMID 1384050. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  17. ^ Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, Sawyer LS, Hendry RM, Dunlop N, Nara PL (1994). "Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody". Science. 266 (5187): 1024–7. doi:10.1126/science.7973652. PMID 7973652. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  18. ^ Yang WP, Green K, Pinz-Sweeney S, Briones AT, Burton DR, Barbas CF (1995). "CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range". J. Mol. Biol. 254 (3): 392–403. doi:10.1006/jmbi.1995.0626. PMID 7490758. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  19. ^ Barbas CF, Hu D, Dunlop N, Sawyer L, Cababa D, Hendry RM, Nara PL, Burton DR (1994). "In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity". Proc. Natl. Acad. Sci. U.S.A. 91 (9): 3809–13. PMC 43671. PMID 8170992. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  20. ^ Barbas CF, Bain JD, Hoekstra DM, Lerner RA (1992). "Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem". Proc. Natl. Acad. Sci. U.S.A. 89 (10): 4457–61. PMC 49101. PMID 1584777. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  21. ^ Barbas CF, Languino LR, Smith JW (1993). "High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site". Proc. Natl. Acad. Sci. U.S.A. 90 (21): 10003–7. doi:10.1073/pnas.90.21.10003. PMC 47701. PMID 7694276. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  22. ^ Barbas CF, Wagner J (1995). "Synthetic Human Antibodies: Selecting and Evolving Functional Proteins". Methods. 8 (2): 94–103. doi:10.1006/meth.1995.9997. {{cite journal}}: Unknown parameter |month= ignored (help)
  23. ^ Barbas CF (1995). "Synthetic human antibodies". Nat. Med. 1 (8): 837–9. doi:10.1038/nm0895-837. PMID 7585190. {{cite journal}}: Unknown parameter |month= ignored (help)
  24. ^ Lawrence S (2007). "Billion dollar babies--biotech drugs as blockbusters". Nat. Biotechnol. 25 (4): 380–2. doi:10.1038/nbt0407-380. PMID 17420735. {{cite journal}}: Unknown parameter |month= ignored (help)
  25. ^ Cambridge Antibody: Sales update | Company Announcements | Telegraph
  26. ^ Huang J, Ru B, Dai P (2011). "Bioinformatics resources and tools for phage display". Molecules. 16 (1): 694–709. doi:10.3390/molecules16010694. PMID 21245805.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  27. ^ Huang J, Ru B, Zhu P, Nie F, Yang J, Wang X, Dai P, Lin H, Guo FB, Rao N (2012). "MimoDB 2.0: a mimotope database and beyond". Nucleic Acids Res. 40 (Database issue): D271–7. doi:10.1093/nar/gkr922. PMC 3245166. PMID 22053087. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  28. ^ Huang J, Ru B, Li S, Lin H, Guo FB (2010). "SAROTUP: scanner and reporter of target-unrelated peptides". J. Biomed. Biotechnol. 2010: 101932. doi:10.1155/2010/101932. PMC 2842971. PMID 20339521.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)

Further reading

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