Editing Phage display

{{Short description|Biological technique to evolve proteins using bacteriophages}}
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{{Technical|date=October 2018}}
[[File:Phage display.png|right|thumb|400px|Phage display cycle. 1) fusion proteins for a viral coat protein + the gene to be evolved (typically an antibody fragment) are expressed in bacteriophage. 2) the library of phage are washed over an immobilised target. 3) the remaining high-affinity binders are used to infect bacteria. 4) the genes encoding the high-affinity binders are isolated. 5) those genes may have random mutations introduced and used to perform another round of evolution. The selection and amplification steps can be performed multiple times at greater stringency to isolate higher-affinity binders.]]
'''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 ([[viruses]] that infect [[bacteria]]) to connect proteins with the [[genetic information]] that [[code|encodes]] them.<ref name="Smith_1985">{{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 = 1315–7 |date=June 1985 | pmid = 4001944 | doi = 10.1126/science.4001944 |bibcode = 1985Sci...228.1315S }}</ref> In this technique, a gene encoding a protein of interest is inserted into a phage [[viral coat protein|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]]. The proteins that the phages are displaying can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those of other molecules. In this way, large [[protein fragment library|libraries of proteins]] can 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 [[Filamentous bacteriophage fd|fd]] [[filamentous phage]],<ref name="pmid11848876">{{cite journal |vauthors=Smith GP, Petrenko VA | title = Phage Display | journal = Chem. Rev. | volume = 97 | issue = 2 | pages = 391–410 |date=April 1997 | pmid = 11848876 | doi = 10.1021/cr960065d  }}</ref><ref name="pmid16277371">{{cite journal |vauthors=Kehoe JW, Kay BK | title = Filamentous phage display in the new millennium | journal = Chem. Rev. | volume = 105 | issue = 11 | pages = 4056–72 |date=November 2005 | pmid = 16277371 | doi = 10.1021/cr000261r }}</ref> though [[Enterobacteria phage T4|T4]],<ref name="Malys_2002">{{cite journal |vauthors=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.

==History==
Phage display was first described by [[George Smith (chemist)|George P. Smith]] in 1985, when he demonstrated the display of peptides on [[filamentous phage]] (long, thin viruses that infect bacteria) by [[Fusion protein|fusing]] the virus's [[Capsid|capsid protein]] to one [[peptide]] out of a collection of peptide sequences.<ref name="Smith_1985" /> This displayed the different peptides on the outer surfaces of the collection of viral clones, where the screening step of the process isolated the peptides with the highest binding affinity. In 1988, Stephen Parmley and George Smith described [[biopanning]] for affinity selection and demonstrated that recursive rounds of selection could enrich for clones present at 1 in a billion or less.<ref>{{cite journal|doi=10.1016/0378-1119(88)90495-7 | volume=73 | title=Antibody-selectable filamentous fd phage vectors: affinity purification of target genes | year=1988 | journal=Gene | pages=305–318 | vauthors=Parmley SF, Smith GP| issue=2 | pmid=3149606 }}</ref> In 1990, Jamie Scott and George Smith described creation of large random peptide libraries displayed on filamentous phage.<ref>{{Cite journal |doi = 10.1126/science.1696028|pmid = 1696028|bibcode = 1990Sci...249..386S|title = Searching for peptide ligands with an epitope library|year = 1990|last1 = Scott|first1 = J.|last2 = Smith|first2 = G.|journal = Science|volume = 249|issue = 4967|pages = 386–390}}</ref> Phage display technology was further developed and improved by groups at the [[Laboratory of Molecular Biology]] with [[Greg Winter]] and [[John McCafferty]], The [[Scripps Research Institute]] with Richard Lerner and Carlos Barbas and the [[German Cancer Research Center]] with Frank Breitling and [[Stefan Dübel]] for display of proteins such as [[antibodies]] for [[therapeutic]] [[protein engineering]]. Smith and Winter were awarded a half share of the 2018 Nobel Prize in chemistry for their contribution to developing phage display.<ref>{{Cite web|url=https://www.nobelprize.org/prizes/chemistry/2018/summary/|title=The Nobel Prize in Chemistry 2018|website=NobelPrize.org|language=en-US|access-date=2018-10-03}}</ref> A patent by George Pieczenik claiming priority from 1985 also describes the generation of peptide libraries.<ref name="US_5866363">{{ cite patent | country = US | number = 5866363  | status = patent | title = Method and means for sorting and identifying biological information | pubdate = 1999-02-02 | fdate = 1991-02-28 | pridate = 1985-08-28 | inventor = Pieczenik G}}</ref>

== 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>

== Applications ==

Applications of phage display 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 the function or the mechanism of the function of that protein may be determined.<ref>[https://web.archive.org/web/20060628072224/http://genome.wellcome.ac.uk/doc_WTD020763.html Explanation of "Protein interaction mapping" from The Wellcome Trust]</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 name="pmid16258189">{{cite journal |vauthors=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 |date=November 2005 | pmid = 16258189 | doi = 10.1385/ABAB:127:2:125 | s2cid = 45243314 }}</ref><ref name="pmid15913550">{{cite journal |vauthors=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 |date=July 2005 | pmid = 15913550 | doi = 10.1016/j.bbrc.2005.05.028 }}</ref><ref name="pmid15863836">{{cite journal |vauthors=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 |date=July 2005 | pmid = 15863836 | doi = 10.1194/jlr.M500048-JLR200  | doi-access = free }}</ref> The technique is also used to determine [[Tumor antigen|tumour antigens]] (for use in diagnosis and therapeutic targeting)<ref name="Hufton_1999">{{cite journal |vauthors=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 |date=December 1999 | pmid = 10648926 | doi = 10.1016/S0022-1759(99)00139-8 }}</ref> and in searching for [[protein-DNA interaction]]s<ref name="Gommans_2005">{{cite journal |vauthors=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 |date=December 2005 | pmid = 16253273 | doi = 10.1016/j.jmb.2005.06.082 }}</ref> using specially-constructed DNA libraries with randomised segments. Recently, phage display has also been used in the context of cancer treatments - such as the adoptive cell transfer approach.<ref name=":0">{{cite web|title=CAR T Cells: Engineering Patients' Immune Cells to Treat Their Cancers|url=https://www.cancer.gov/about-cancer/treatment/research/car-t-cells|website=National Cancer Institute|access-date=9 February 2018|date=2013-12-06}}</ref> In these cases, phage display is used to create and select synthetic antibodies that target tumour surface proteins.<ref name=":0" /> These are made into synthetic receptors for T-Cells collected from the patient that are used to combat the disease.<ref>{{cite journal | vauthors = Løset GÅ, Berntzen G, Frigstad T, Pollmann S, Gunnarsen KS, Sandlie I | title = Phage Display Engineered T Cell Receptors as Tools for the Study of Tumor Peptide-MHC Interactions | journal = Frontiers in Oncology | volume = 4 | issue = 378 | pages = 378 | date = 12 January 2015 | pmid = 25629004 | pmc = 4290511 | doi = 10.3389/fonc.2014.00378 | doi-access = free }}</ref>

Competing methods for [[directed evolution|''in vitro'' protein evolution]] include [[yeast display]], [[bacterial display]], [[ribosome display]], and [[mRNA display]].{{cn|date=October 2022}}

=== Antibody maturation ''in vitro'' ===
The invention of [[antibody]] phage display revolutionised antibody drug discovery. Initial work was done by laboratories at the [[MRC Laboratory of Molecular Biology]] ([[Greg Winter]] and [[John McCafferty]]), the [[Scripps Research Institute]] (Richard Lerner and Carlos F. Barbas) and the [[German Cancer Research Centre]] (Frank Breitling and Stefan Dübel).<ref name="pmid2247164">{{cite journal | vauthors = 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 | date = December 1990 | pmid = 2247164 | doi = 10.1038/348552a0 | bibcode = 1990Natur.348..552M | s2cid = 4258014 | author-link4 = David Chiswell | author-link1 = John McCafferty | author-link3 = Greg Winter }}</ref><ref name="isbn0-87969-740-7">{{cite book | vauthors = 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 | isbn = 978-0-87969-740-2 }}</ref><ref>{{cite journal | vauthors = Breitling F, Dübel S, Seehaus T, Klewinghaus I, Little M | title = A surface expression vector for antibody screening | journal = Gene | volume = 104 | issue = 2 | pages = 147–53 | date = August 1991 | pmid = 1916287 | doi = 10.1016/0378-1119(91)90244-6 }}</ref> In 1991, The Scripps group reported the first display and selection of human antibodies on phage.<ref name="pmid1896445">{{cite journal | vauthors = Barbas CF, Kang AS, Lerner RA, Benkovic SJ | title = Assembly of combinatorial antibody libraries on phage surfaces: the gene III site | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 88 | issue = 18 | pages = 7978–82 | date = September 1991 | pmid = 1896445 | pmc = 52428 | doi = 10.1073/pnas.88.18.7978 | bibcode = 1991PNAS...88.7978B | doi-access = free }}</ref> This initial study described the rapid isolation of human antibody [[Fragment antigen-binding|Fab]] 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 | vauthors = 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 = Proceedings of the National Academy of Sciences of the United States of America | volume = 88 | issue = 22 | pages = 10134–7 | date = November 1991 | pmid = 1719545 | pmc = 52882 | doi = 10.1073/pnas.88.22.10134 | bibcode = 1991PNAS...8810134B | doi-access = free }}</ref><ref name="pmid1384050">{{cite journal | vauthors = 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 = Proceedings of the National Academy of Sciences of the United States of America | volume = 89 | issue = 19 | pages = 9339–43 | date = October 1992 | pmid = 1384050 | pmc = 50122 | doi = 10.1073/pnas.89.19.9339 | bibcode = 1992PNAS...89.9339B | doi-access = free }}</ref><ref name="pmid7973652">{{cite journal | vauthors = 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 | date = November 1994 | pmid = 7973652 | doi = 10.1126/science.7973652 | bibcode = 1994Sci...266.1024B }}</ref><ref name="pmid7490758">{{cite journal | vauthors = 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 = Journal of Molecular Biology | volume = 254 | issue = 3 | pages = 392–403 | date = December 1995 | pmid = 7490758 | doi = 10.1006/jmbi.1995.0626 }}</ref><ref name="pmid8170992">{{cite journal | vauthors = 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 = Proceedings of the National Academy of Sciences of the United States of America | volume = 91 | issue = 9 | pages = 3809–13 | date = April 1994 | pmid = 8170992 | pmc = 43671 | doi = 10.1073/pnas.91.9.3809 | bibcode = 1994PNAS...91.3809B | doi-access = free }}</ref>

Phage display of antibody libraries has become 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 synthetic human antibody libraries, a principle first patented in 1990 by Breitling and coworkers (Patent CA 2035384), thereby allowing human antibodies to be created in vitro from synthetic diversity elements.<ref name="pmid1584777">{{cite journal |vauthors=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 |date=May 1992 | pmid = 1584777 | pmc = 49101 | doi =10.1073/pnas.89.10.4457 | bibcode = 1992PNAS...89.4457B | doi-access = free }}</ref><ref name="pmid7694276">{{cite journal |vauthors=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 |date=November 1993 | pmid = 7694276 | pmc = 47701 | doi = 10.1073/pnas.90.21.10003 |bibcode = 1993PNAS...9010003B | doi-access = free }}</ref><ref name=Barbas_1995>{{cite journal |vauthors=Barbas CF, Wagner J | title = Synthetic Human Antibodies: Selecting and Evolving Functional Proteins | journal = Methods |date=October 1995 | 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 |date=August 1995 | pmid = 7585190 | doi = 10.1038/nm0895-837 | s2cid = 6983649 }}</ref>

Antibody libraries displaying millions of different antibodies on phage are often used in the pharmaceutical industry to isolate highly specific therapeutic antibody leads, for development into antibody drugs primarily as anti-cancer or anti-inflammatory therapeutics. One of the most successful was [[adalimumab]], discovered by [[Cambridge Antibody Technology]] as D2E7 and developed and marketed by [[Abbott Laboratories]]. Adalimumab, 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 |date=April 2007 | pmid = 17420735 | doi = 10.1038/nbt0407-380 | s2cid = 205266758 }}</ref> to achieve annual sales exceeding $1bn.<ref>[https://web.archive.org/web/20110717145527/http://telegraph.uk-wire.com/cgi-bin/articles/200601251501444434X.html Cambridge Antibody: Sales update | Company Announcements | Telegraph<!-- Bot generated title -->]</ref>

== General protocol ==

Below is the sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence:{{cn|date=October 2022}}
# Target proteins or DNA sequences are immobilized 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 phage (i.e. non-binding) than were present in the initial mixture.
# Steps 3 to 5 are optionally repeated one or more times, further enriching the phage library in binding proteins.
# Following further bacterial-based amplification, the DNA within the interacting phage is sequenced to identify the interacting proteins or protein fragments.

== Selection of the coat protein ==

=== Filamentous phages ===

==== pIII ====

pIII is the protein that determines the infectivity of the virion. pIII is composed of three domains (N1, N2 and CT) connected by glycine-rich linkers.<ref name="Lowman_Clackson_2004">{{cite book |vauthors=Lowman HB, Clackson T | title = Phage display: a practical approach | publisher = Oxford University Press | location = Oxford [Oxfordshire] | year = 2004 | pages = 10–11 | isbn = 978-0-19-963873-4 | chapter =  1.3 }}</ref> The N2 domain binds to the F pilus during virion infection freeing the N1 domain which then interacts with a TolA protein on the surface of the bacterium.<ref name="Lowman_Clackson_2004"/> Insertions within this protein are usually added in position 249 (within a linker region between CT and N2), position 198 (within the N2 domain) and at the [[N-terminus]] (inserted between the N-terminal secretion sequence and the N-terminus of pIII).<ref name="Lowman_Clackson_2004"/> However, when using the BamHI site located at position 198 one must be careful of the unpaired Cysteine residue (C201) that could cause problems during phage display if one is using a non-truncated version of pIII.<ref name="Lowman_Clackson_2004"/>

An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid (plasmid derived from [[Ff phages]]) combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences (>100 amino acids)<ref name="pmid10669603">{{cite journal |vauthors=Sidhu SS, Weiss GA, Wells JA | title = High copy display of large proteins on phage for functional selections | journal = J. Mol. Biol. | volume = 296 | issue = 2 | pages = 487–95 |date=February 2000 | pmid = 10669603 | doi = 10.1006/jmbi.1999.3465 }}</ref> and is more tolerant to it than pVIII. However, using pIII as the fusion partner can lead to a decrease in phage infectivity leading to problems such as selection bias caused by difference in phage growth rate<ref name="pmid20583018">{{cite journal | vauthors = Derda R, Tang SK, Whitesides GM | title = Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets | journal = Angew. Chem. Int. Ed. Engl. | volume = 49 | issue = 31 | pages = 5301–4 | date = July 2010 | pmid = 20583018 | pmc = 2963104 | doi = 10.1002/anie.201001143 }}</ref> or even worse, the phage's inability to infect its host.<ref name="Lowman_Clackson_2004"/> Loss of phage infectivity can be avoided by using a phagemid plasmid and a helper phage so that the resultant phage contains both wild type and fusion pIII.<ref name="Lowman_Clackson_2004"/>

cDNA has also been analyzed using pIII via a two complementary leucine zippers system,<ref name="pmid7957259">{{cite journal |vauthors=Crameri R, Jaussi R, Menz G, Blaser K | title = Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction | journal = Eur. J. Biochem. | volume = 226 | issue = 1 | pages = 53–8 |date=November 1994 | pmid = 7957259 | doi = 10.1111/j.1432-1033.1994.00t53.x }}</ref> Direct Interaction Rescue<ref name="pmid7838733">{{cite journal |vauthors=Gramatikoff K, Georgiev O, Schaffner W | title = Direct interaction rescue, a novel filamentous phage technique to study protein-protein interactions | journal = Nucleic Acids Res. | volume = 22 | issue = 25 | pages = 5761–2 |date=December 1994 | pmid = 7838733 | pmc = 310144 | doi = 10.1093/nar/22.25.5761}}</ref> or by adding an 8-10 amino acid linker between the cDNA and pIII at the C-terminus.<ref name="pmid11034335">{{cite journal |vauthors=Fuh G, Sidhu SS | title = Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein | journal = FEBS Lett. | volume = 480 | issue = 2–3 | pages = 231–4 |date=September 2000 | pmid = 11034335 | doi = 10.1016/s0014-5793(00)01946-3| s2cid = 23009887 | doi-access = free | bibcode = 2000FEBSL.480..231F }}</ref>

==== pVIII ====

pVIII is the main coat protein of Ff phages. Peptides are usually fused to the N-terminus of pVIII.<ref name="Lowman_Clackson_2004"/> Usually peptides that can be fused to pVIII are 6-8 amino acids long.<ref name="Lowman_Clackson_2004"/> The size restriction seems to have less to do with structural impediment caused by the added section<ref name="Malik_1998">{{cite journal |vauthors=Malik P, Terry TD, Bellintani F, Perham RN | title = Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase | journal = FEBS Lett. | volume = 436 | issue = 2 | pages = 263–6 |date=October 1998 | pmid = 9781692 | doi = 10.1016/s0014-5793(98)01140-5| s2cid = 19331069 | doi-access = free | bibcode = 1998FEBSL.436..263M }}</ref> and more to do with the size exclusion caused by pIV during coat protein export.<ref name="Malik_1998"/> Since there are around 2700 copies of the protein on a typical phages, it is more likely that the protein of interest will be expressed polyvalently even if a phagemid is used.<ref name="Lowman_Clackson_2004"/> This makes the use of this protein unfavorable for the discovery of high affinity binding partners.<ref name="Lowman_Clackson_2004"/>

To overcome the size problem of pVIII, artificial coat proteins have been designed.<ref name="Weiss_Sidhu_2000">{{cite journal |vauthors=Weiss GA, Sidhu SS | title = Design and evolution of artificial M13 coat proteins | journal = J. Mol. Biol. | volume = 300 | issue = 1 | pages = 213–9 |date=June 2000 | pmid = 10864510 | doi = 10.1006/jmbi.2000.3845 }}</ref> An example is Weiss and Sidhu's inverted artificial coat protein (ACP) which allows the display of large proteins at the C-terminus.<ref name="Weiss_Sidhu_2000"/> The ACP's could display a protein of 20kDa, however, only at low levels (mostly only monovalently).<ref name="Weiss_Sidhu_2000"/>

==== pVI ====

pVI has been widely used for the display of cDNA libraries.<ref name="Lowman_Clackson_2004"/> The display of cDNA libraries via phage display is an attractive alternative to the yeast-2-hybrid method for the discovery of interacting proteins and peptides due to its high throughput capability.<ref name="Lowman_Clackson_2004"/> pVI has been used preferentially to pVIII and pIII for the expression of cDNA libraries because one can add the protein of interest to the C-terminus of pVI without greatly affecting pVI's role in phage assembly. This means that the stop codon in the cDNA is no longer an issue.<ref name="pmid9634780">{{cite journal |vauthors=Jespers LS, Messens JH, De Keyser A, Eeckhout D, Van den Brande I, Gansemans YG, Lauwereys MJ, Vlasuk GP, Stanssens PE | title = Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI | journal = Bio/Technology | volume = 13 | issue = 4 | pages = 378–82 |date=April 1995 | pmid = 9634780 | doi = 10.1038/nbt0495-378| s2cid = 6171262 }}</ref> However, phage display of cDNA is always limited by the inability of most prokaryotes in producing post-translational modifications present in eukaryotic cells or by the misfolding of multi-domain proteins.

While pVI has been useful for the analysis of cDNA libraries, pIII and pVIII remain the most utilized coat proteins for phage display.<ref name="Lowman_Clackson_2004"/>

==== pVII and pIX ====

In an experiment in 1995, display of Glutathione S-transferase was attempted on both pVII and pIX and failed.<ref name="pmid7616570">{{cite journal |vauthors=Endemann H, Model P | title = Location of filamentous phage minor coat proteins in phage and in infected cells | journal = J. Mol. Biol. | volume = 250 | issue = 4 | pages = 496–506 |date=July 1995 | pmid = 7616570 | doi =  10.1006/jmbi.1995.0393}}</ref> However, phage display of this protein was completed successfully after the addition of a periplasmic signal sequence (pelB or ompA) on the N-terminus.<ref name="pmid10339535">{{cite journal |vauthors=Gao C, Mao S, Lo CH, Wirsching P, Lerner RA, Janda KD | title = Making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 96 | issue = 11 | pages = 6025–30 |date=May 1999 | pmid = 10339535 | pmc = 26829 | doi =  10.1073/pnas.96.11.6025|bibcode = 1999PNAS...96.6025G | doi-access = free }}</ref> In a recent study, it has been shown that AviTag, FLAG and His could be displayed on pVII without the need of a signal sequence.  Then the expression of single chain Fv's (scFv), and single chain T cell receptors (scTCR) were expressed both with and without the signal sequence.<ref name="Løset_2011">{{cite journal |vauthors=Løset GÅ, Roos N, Bogen B, Sandlie I | title = Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage | journal = PLOS ONE | volume = 6 | issue = 2 | pages = e17433 | year = 2011 | pmid = 21390283 | pmc = 3044770 | doi = 10.1371/journal.pone.0017433 |bibcode = 2011PLoSO...617433L | doi-access = free }}</ref>

PelB (an amino acid signal sequence that targets the protein to the periplasm where a signal peptidase then cleaves off PelB) improved the phage display level when compared to pVII and pIX fusions without the signal sequence. However, this led to the incorporation of more helper phage genomes rather than phagemid genomes. In all cases, phage display levels were lower than using pIII fusion. However, lower display might be more favorable for the selection of binders due to lower display being closer to true monovalent display. In five out of six occasions, pVII and pIX fusions without pelB was more efficient than pIII fusions in affinity selection assays. The paper even goes on to state that pVII and pIX display platforms may outperform pIII in the long run.<ref name="Løset_2011"/>

The use of pVII and pIX instead of pIII might also be an advantage because virion rescue may be undertaken without breaking the virion-antigen bond if the pIII used is wild type. Instead, one could cleave in a section between the bead and the antigen to elute. Since the pIII is intact it does not matter whether the antigen remains bound to the phage.<ref name="Løset_2011"/>

=== T7 phages ===

The issue of using Ff phages for phage display is that they require the protein of interest to be translocated across the bacterial inner membrane before they are assembled into the phage.<ref name="Danner_Belasco_2001">{{cite journal |vauthors=Danner S, Belasco JG | title = T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 98 | issue = 23 | pages = 12954–9 |date=November 2001 | pmid = 11606722 | pmc = 60806 | doi = 10.1073/pnas.211439598 |bibcode = 2001PNAS...9812954D | doi-access = free }}</ref> Some proteins cannot undergo this process and therefore cannot be displayed on the surface of Ff phages. In these cases, T7 phage display is used instead.<ref name="Danner_Belasco_2001"/> In T7 phage display, the protein to be displayed is attached to the C-terminus of the gene 10 capsid protein of T7.<ref name="Danner_Belasco_2001"/>

The disadvantage of using T7 is that the size of the protein that can be expressed on the surface is limited to shorter peptides because large changes to the T7 genome cannot be accommodated like it is in M13 where the phage just makes its coat longer to fit the larger genome within it. However, it can be useful for the production of a large protein library for scFV selection where the scFV is expressed on an M13 phage and the antigens are expressed on the surface of the T7 phage.<ref name="pmid11687245">{{cite journal |vauthors=Castillo J, Goodson B, Winter J | title = T7 displayed peptides as targets for selecting peptide specific scFvs from M13 scFv display libraries | journal = J. Immunol. Methods | volume = 257 | issue = 1–2 | pages = 117–22 |date=November 2001 | pmid = 11687245 | doi =  10.1016/s0022-1759(01)00454-9}}</ref>

== Bioinformatics resources and tools ==

Databases and computational tools for [[mimotope]]s have been an important part of phage display study.<ref name="pmid21245805">{{cite journal |vauthors=Huang J, Ru B, Dai P | title = Bioinformatics resources and tools for phage display | journal = Molecules | volume = 16 | issue = 1 | pages = 694–709 | year = 2011 | pmid = 21245805 | doi = 10.3390/molecules16010694 | pmc = 6259106 | doi-access = free }}</ref> Databases,<ref name="pmid22053087">{{cite journal |vauthors=Huang J, 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 Res. | volume = 40 | issue = Database issue | pages = D271–7 |date=January 2012 | pmid = 22053087 | pmc = 3245166 | doi = 10.1093/nar/gkr922 }}</ref> programs and web servers<ref name="PMC2808184">{{cite journal |vauthors=Negi SS, Braun W | title = Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences | journal = Bioinform Biol Insights | volume = 3 |pages = 71–81 | year = 2009 | pmid = 20140073 | pmc=2808184| doi = 10.4137/BBI.S2745 }}</ref> have been widely used to exclude target-unrelated peptides,<ref name="pmid20339521">{{cite journal |vauthors=Huang J, Ru B, Li S, Lin H, Guo FB | title = SAROTUP: scanner and reporter of target-unrelated peptides | journal = J. Biomed. Biotechnol. | volume = 2010 | page = 101932 | year = 2010 | pmid = 20339521 | pmc = 2842971 | doi = 10.1155/2010/101932 | doi-access = free }}</ref> characterize small molecules-protein interactions and map protein-protein interactions. Users can use three dimensional structure of a protein and the peptides selected from phage display experiment to map conformational epitopes. Some of the fast and efficient computational methods are available online.<ref name="PMC2808184"/>

== See also ==
* [[Directed evolution]]
* [[protein–protein interactions]]
* [[PelB leader sequence]]
Competing techniques:
* [[Two-hybrid system]]
* [[mRNA display]]
* [[Ribosome display]]
* [[Yeast display]]

== References ==
{{Reflist|35em}}

==Further reading==
{{refbegin}}
* {{cite journal | vauthors = Ledsgaard L, Kilstrup M, Karatt-Vellatt A, McCafferty J, Laustsen AH | title = Basics of antibody phage display technology | journal = Toxins | volume = 10 | issue = 6 | pages = 236 | year = 2018 | pmid = 29890762 | pmc = 6024766 | doi = 10.3390/toxins10060236 | url = https://orbit.dtu.dk/files/150474594/toxins_10_00236.pdf | doi-access = free }}
* [http://nobelprize.org/nobelfoundation/symposia/chemistry/ncs-2001-2/abstract-smith.html Selection Versus Design in Chemical Engineering]
* [https://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&catid=4&sobi2Id=331&Itemid=81 The ETH-2 human antibody phage library] {{Webarchive|url=https://web.archive.org/web/20110715124631/https://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&catid=4&sobi2Id=331&Itemid=81 |date=2011-07-15 }}
* {{cite book |vauthors=Sidhu SS, Lowman HB, Cunningham BC, Wells JA | chapter = Phage display for selection of novel binding peptides | title = Applications of Chimeric Genes and Hybrid Proteins - Part C: Protein-Protein Interactions and Genomics | volume = 328 | pages = 333–63 | year = 2000 | pmid = 11075354 | doi = 10.1016/S0076-6879(00)28406-1 | series = Methods in Enzymology | isbn = 9780121822293 }}
{{refend}}

== External links ==
{{Library resources box
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{{Protein methods}}
{{DEFAULTSORT:Phage Display}}
[[Category:Molecular biology]]
[[Category:Bacteriophages]]
[[Category:Microbiology]]
[[Category:Protein–protein interaction assays]]
[[Category:Display techniques]]