Plant Molecular Biology 50: 837–854, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Phage display: practicalities and prospects

William G.T. WillatsDepartment of Biochemistry and Molecular Biology; University of Leeds, Woodhouse Lane, LS2 9JT, UK(Tel: +44 (0)113 3433168; Fax: +44 (0)113 3433144; E-mail: w.g.t.willats@leeds.ac.uk)

Accepted 20 August 2002

Key words: antibody microarrays, immunomodulation, molecular evolution, Phage display, protein interactions,recombinant antibodies

Abstract

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles.Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequenceencoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variantnucleotide sequences may be converted into populations of variant peptides and proteins which may be screened fordesired properties. It is now some seventeen years since the first demonstration of the feasibility of this technologyand the intervening years have seen an explosion in its applications. This review discusses the major uses ofphage display including its use for elucidating protein interactions, molecular evolution and for the production ofrecombinant antibodies.

Abbreviations: scFv – single chain variable fragment of an antibody; dsFv – disulphide stabilised scFv; Fab –antigen binding antibody fragment; PCR – polymerase chain reaction; RG II – rhamnogalacturonan II; CDR –complimentarity determining region; VH – variable region of an antibody heavy chain; VL – variable region of anantibody light chain; HG – homogalacturonan; AceHG – acetylated HG; ELISA – enzyme linked immunosorbentassay; GFP – green fluorescent protein.

Introduction

Phage display technology has had a major impact onimmunology, cell biology, drug discovery and phar-macology and is increasingly gaining importance inplant science. The aim of this review is to providea practical survey of the principles and applicationsof phage display. The emphasis will be on the rela-tive merits of this technology for addressing diversebiological problems and the practicalities of what is in-volved, rather than a detailed exposition of moleculartechnique or a comprehensive review of the literature.

Phage display is an extremely powerful tool forselecting peptides or proteins with specific bindingproperties from vast numbers of variants. Its utilitylies principally in generating molecular probes againstspecific targets and for the analysis and manipulationof protein/ligand interactions. Put at its most simple,phage display is the expression of peptides, proteins

or antibody fragments at the surface of phage par-ticles (Smith, 1985; Winter et al., 1994; Kay andHoess,1996). This is accomplished by the incorpora-tion of the nucleotide sequence encoding the proteinto be displayed into a phage or phagemid genome asa fusion to a gene encoding a phage coat protein. Thisfusion ensures that as phage particles are assembled,the protein to be displayed is presented at the surfaceof the mature phage, while the sequence encoding it iscontained within the same phage particle (Figure 1aand 1b). This physical link between the phenotypeand genotype of the expressed protein and the replica-tive capacity of phage are the structural elements thatunderpin all phage display technology (Figure 1b).Using phage display, libraries of variant nucleotidesequences with diversities of millions or billions maybe converted into populations of displayed variant pro-teins which can then be conveniently screened for

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Figure 1. The principle of phage display. (a) The simplified hypothetical bacteriophage shown has a genome that contains an origin ofreplication (Or) and genes (g1 and g2) that encode two types of coat protein - p1 and p2, respectively. A foreign protein pχ , that is encodedby gene gχ , may be displayed at the phage surface by the fusion of gχ to one of the phage coat protein genes. The number of copies of pχ

displayed is related to which phage coat protein (p1 or p2) is chosen as a fusion partner. (b) This principle can be applied to the expression ofnatural or random peptides, protein domains or whole proteins and antibody fragments. (c) Using phage display, a library of variant nucleotidesequences can be converted into a library of variant peptides or proteins. The phage display library may then be screened in order to isolatephage displaying peptides or proteins with desired properties.

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desirable properties (Figure 1c). Screening of phagedisplay libraries is usually accomplished by an affin-ity selection (or bio-panning) process during whichphage populations are exposed to targets in orderto selectively capture binding phage (Hoogenboom,1997; Hoogenboom et al., 1998; Sparks et al., 1996).Throughout successive rounds of binding, washing,elution and amplification, the originally very diversephage population is increasingly enriched with phagewith a propensity to bind to the target in question. Ul-timately, monoclonal phage populations with desiredspecificities can be selected. This procedure of DNAmanipulation to create a library of variants, packaginginto phage, and subsequent bio-panning is the basicprotocol for all phage display and has been coinedthe ‘phage display cycle’ (Hoogenboom et al., 1998)(Figure 2). Because the genotype of each protein phe-notype is carried within phage particles, once proteinsof interest have been isolated the sequence encodingthem can be readily determined and altered in order tomanipulate or refine binding properties.

Phage display WWW resources

A wealth of information and physical resources relat-ing to phage display is now available via the WorldWide Web. Table 1 is a list of the URLs for someof the major sources of phage display libraries andinformation.

Why use phage display?

As a system for the high throughput analysis of proteininteractions phage display is complimentary to, ratherthan a substitute for, other methods such as yeast hy-brid systems (Drees, 1999; Mendelsohn and Brent,1999; Uetz, 2001) (see also pages X – X of this issue)and each have their advantages and limitations. Oneadvantage of phage display is the enormous diversityof variant proteins that can be represented. For exam-ple, phage display antibody libraries with diversitiesas high as 1010 are routinely constructed (Hoogen-boom et al., 1998) (see also Section 7). Phage displayis highly flexible and selection may be performed invivo or in vitro. (Johns et al., 2000; Sparks et al.,1996; McCafferty and Johnson, 1996). In vitro selec-tion enables phage displayed proteins to be screenednot only against a wide range of biological targets butalso inorganic ones (Whaley et al., 2000). Yeast hybridsystems have the potential advantage that protein in-teractions are assessed under physiological conditions

– but only a limited range of conditions are avail-able, namely those within yeast cells (Drees, 1999). Incontrast, phage display screening formats can be read-ily modified to manipulate selection conditions andstringencies (Watters et al., 1997; Rodi et al., 2001;Hoogenboom et al., 1998). Both yeast hybrid systemsand phage display provide a means of rapidly screen-ing large numbers of proteins against potential bindingpartners but phage display has the higher throughput.As a rough guide, billions of clones can be screenedwithin a week using phage display while with yeasttwo-hybrids millions of clones can be screened intwo to four weeks (Rodi et al., 2001). An attractiveaspect of phage display is that provided appropriatelibraries can be obtained (Table 1), the technique issimple, cheap, rapid to set up and requires no specialequipment.

Phage Display vehicles

The two key physical elements of phage displayare the libraries of nucleotide sequences encodingpeptides or proteins (e.g. gene fragments, randomoligonucleotides, cDNA populations or antibody genesequences) and the phage vehicles on which these se-quences are expressed. The simplest way of achievingthe expression of a foreign protein is to simply cre-ate a fusion between the nucleotide sequence to beexpressed and a coat protein gene within the viralgenome (Figure 3a). Using this direct approach all thecopies of the chosen coat protein become fusion pro-teins (Winter et al., 1994). This can be advantageousin terms of numbers of expressed foreign proteins butif the functionality of the chosen coat protein is com-promised by the fusion then phage viability may beaffected, especially since no wild type versions of thecoat protein are retained. This is avoided if hybridphage are produced in which some versions of a givencoat protein are wild type and some are fused to a for-eign protein (Figure 3b and 3c). In some hybrid phagesystems the gene fusion is an additional element of thephage genome so that a wild type copy of the coatprotein gene is retained and phage particles expressboth wild type and fusion proteins (Figure 3b) (Sidhu,2001). Alternatively, hybrid phage may be created us-ing a phagemid-based system and this approach hasbeen widely adopted (Figure 3c). Sequences encod-ing fusion proteins are carried by phagemids (plasmidswith a phage origin of replication) while the majorityof the genes required for the formation of phage parti-

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Figure 2. The phage display cycle (a) A library of variant DNA sequences encoding peptides or proteins is created and (b) cloned into phage orphagemid genomes as fusions to a coat protein gene (see also Figure 3). (c) The phage library displaying variant peptides or proteins is exposedto target molecules and phage with appropriate specificity are captured. (d) Non binding phage are washed off – although some non-specificbinding may also occur. (e) Bound phage are eluted by conditions that disrupt the interaction between the displayed peptide or protein and thetarget. (f) Eluted phage are infected into host bacterial cells and thereby amplified. (g) This amplified phage population is in effect a secondarylibrary that is greatly enriched in phage displaying peptides or proteins that bind to the target. If the bio-panning steps (c) to (f) are repeatedthe phage populated becomes less and less diverse as the population becomes more and more enriched in the limited number of variants withbinding capacity. (h) After several (usually three to five) rounds of bio-panning monoclonal phage populations may be selected and analysedindividually.

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Table 1. Phage display resources on the WWW.

Source URL Comments

Smith Lab. University of Missouri www.biosci.missouri.edu/smithgp/Pha-geDisplayWebsite/ PhageDisplayWebsite-Index.html

A source of peptide phage display librariesand associated information

MRC Centre for Protein Engineer-ing. The Winter Group home page.

www.mrc-cpe.cam.ac.uk/∼phage/ The Winter group has previously providedsynthetic antibody libraries. Although notdistributed at present, copies of these li-braries may be obtained from existing users.This site also contains useful phage displayinformation.

S. Dübel, at the University of Hei-delberg, Molecular Genetics

www.mgen.uni-heidelberg.de/SD/SDscFvSite.html

A comprehensive source of recombinant an-tibody resources

The Queen’s University of Belfast www.qub.ac.uk/bb/awpage/faq.htm Extensive information about obtaining phagedisplay libraries and associated reagents.General information about phage displayprotocols

New England BioLabs www.neb.com/neb/frame_cat.html Information about the PhDTM phage displaypeptide libraries as well as useful generalphage display information

MRC Centre for Protein Engineer-ing. V Base (MRC)

www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html

A database of human antibody genes

Philipps-Universität Marburg http://aximt1.imt.uni-marburg.de/ rek/AEPphage.html

Phage display and general filamentous phageinformation

University of Nijmegen http://baserv.uci.kun.nl/ jraats/links1.html Comprehensive phage display links

cles are carried by helper phage that are co-infectedtogether with phagemids into host bacteria (Sidhu,2001) (Figure 3c).

Hybrid phage systems have the potential disad-vantage that the average number of displayed fusionproteins is reduced because of competition for incor-poration into the phage particle between wild type andfusion coat proteins (Winter et al., 1994; McCafferty,1996). However, low valency can be used as a strategyto select for high avidity binders during bio-panning.If coat protein functionality is not completely com-promised by fusion to a foreign protein, then valencycan be increased in phagemid systems by the use ofmodified helper phage (such as M13�gIII) that lackthe gene for the chosen coat protein (Winter et al.,1994; Rondot, 2001; Griffiths et al., 1993). More-over, the choice of coat protein fusion partners hasbeen extended recently by the development of newmutant variants of coat proteins and even completelyartificial coat proteins (Sidhu, 2001). The number ofexpressed proteins therefore depends on the coat pro-tein chosen as a fusion partner, the display systemused (phage or phagemid) and, if a phagemid system

is used, the choice of helper phage. A refinement ofsome phage display systems is the insertion of an am-ber stop codon between the sequences encoding thecoat protein and the displayed foreign protein. Thisallows a soluble (i.e. non-phage bound) version of theforeign protein to be produced if the phage are propa-gated in an appropriate non-suppressing strain of hostbacteria (Winter et al., 1994). Peptide tags such as c-myc and poly-histidine are routinely incorporated intodisplayed proteins for ease of subsequent purificationand detection.

Many types of phage have been used as vehi-cles for phage display including Ff filamentous phage,Lambda and T7 (Rodi and Makowski, 1999; Dannerand Belasco, 2001). Each of these has advantages anddisadvantages with respect to each particular applica-tion. The Ff phage family (M13 and its close relativesfd and fl) are excellent cloning vehicles because theirsize is not constrained by the DNA contained withinthem. The insertion of foreign sequences within theirgenome is accommodated simply by the assembly oflonger phage particles. On the other hand, the non-lytic propagation mechanism of Ff phage requires that

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Figure 3. Phage display formats (a)–(c) Strategies for the expression of proteins at the surface of a simplified hypothetical bacteriophage (seealso Figure 1). (a) The simplest format for the expression of a peptide or protein is to fuse the gene (gχ ) encoding the foreign protein (pχ )to one of the phage coat protein genes (e.g., g1) (see also Figure 1). This strategy produces phage particles in which all the copies of chosenphage coat protein are fusion proteins (p1/pχ ). (b) Hybrid phage may be created by incorporating the gene fusion (g1/gχ ) as an additionalelement in the phage genome. With this arrangement, two versions of the phage coat protein chosen as the fusion partner are encoded - one bythe native gene (p1) and one by the fusion gene (p1/pχ ). As phage particles are assembled both p1 and p1/pχ are incorporated into the phagecoat. (c) Phagemid based systems are also widely used to construct hybrid phage. However, instead of being present on a single genome, thegenes encoding wild type coat protein and fused protein are carried by helper phage and phagemid respectively. Host bacteria contain bothphagemid and helper phage DNA and both genomes contribute to the synthesis of hybrid phage particles. (d) M13 bacteriophage are widelyused as vehicles for phage display. The pIII coat protein can be used as a fusion partner for a limited number (maximum of five) of proteinswhile thousands of proteins can be expressed at the phage surface if pVIII is used as a fusion partner. The approximate number of copies ofeach M13 coat protein is indicated.

the all the components of the phage coat be exportedthrough the bacterial inner membrane prior to theassembly of the mature phage particle. As a conse-quence, only proteins that are capable of withstandingthis export may be displayed (Danner and Belasco,2001). This limitation may be avoided by using thelytic phage Lambda and T7, in which capsid assemblyoccurs entirely in the cytoplasm prior to cell lysis. Fur-thermore, recent studies have shown that unlike T7,Lambda phage can tolerate the display of relative large

proteins at high density (Zucconi et al., 2001). In somecases it may even be advantageous to combine dif-ferent phage types in one experiment. This approachwas used by Castillo et al. (2001) in order to selectanti-peptide single chain antibody fragments (scFvs).Whilst the peptide targets were displayed on T7, thescFvs were selected from an M13 display library.

Despite some limitations, the Ff bacteriophageprovide a robust and highly flexible platform for dis-play and have been widely adopted. These long (about

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1 µm) phage particles consist of single stranded DNApackaged into a coat consisting of five different typesof coat protein, all of which have been used for thedisplay of foreign proteins (Figure 3d) (Sidhu, 2001;Hoogenboom et al., 1998; Rodi and Makowski, 1999).Each of the coat proteins have their relative merits asfusion partners with respect to the number of fusionproteins displayed per phage, the effects of expressedfusion proteins on phage viability, and stability of thefusion proteins (Sidhu, 2001; Rodi and Makowski,1999). In general terms, large numbers of smaller pro-teins may be displayed if pVIII is chosen as a fusionpartner, whilst pIII is a suitable partner for smallernumbers of larger proteins.

Finding the needle in the haystack: screeningphage display libraries

Once a phage display library has been constructed oracquired the task is to screen the library in such a waythat the original very high diversity of the library isreduced to a manageable number of clones which canthen be analysed in detail. Most screening proceduresare based on affinity selection and involve the follow-ing fundamental steps: 1, A library is amplified andphage particles produced; 2, phage particles are ex-posed to a target for which a binding protein is sought;3, non-binding phage are removed by washing and4, binding phage are eluted, infected into host bac-teria and thereby amplified. These bio-panning roundsare then repeated, typically three to six times. In thefollowing sub-sections some general considerationsinvolved in the various steps of phage display libraryscreening are discussed.

Library amplification

Although libraries with very high diversities are avail-able, some expressed sequences are incompatible withphage propagation, whilst others are highly suscepti-ble to proteolysis during propagation. These factorsimpose constraints on effective diversity and it istherefore desirable to start with a library that is asdiverse as possible (Sparks et al., 1996). The possibil-ity that some expressed sequences may be somewhatdeleterious to phage propagation can be militated tosome extent by including a growth step in each pan-ning round that creates less competitive growth condi-tions, for example by growing on solid media ratherthan exclusively in liquid culture. It is also important

to empirically check the diversity of libraries beforestarting any screen because of the possibility that in-stabilities in libraries can lead to loss of inserts. Aquick check of diversity can be made by simply platingout a representative portion of the library, selecting anumber of individual clones and then using PCR tocheck what proportion of clones contain inserts.

Bringing phage and targets together

One of the strengths of phage display is that screen-ing protocols can readily be tailored to the particularrequirements of many different target molecules. Thesimplest and most widely used approach is to immo-bilise target molecules to a support and then to exposesolutions containing phage to the immobilised target.Many variations to this theme have been successfullyused including immobilisation onto coated tubes orplates, within columns or on the surface of magneticbeads. Immobilisation of many bio-molecules canbe achieved by passive adsorption onto polystyrenetubes with an appropriate surface modification such asMaxiSorpTM (Nissim et al., 1994). Passive adsorptionhas the convenience that a wide range of moleculescan be immobilised without any prior treatment. How-ever, passive adsorption relies on establishing a largenumber of relatively weak bonds between target andsupport which can result in the immobilised mole-cule being forced out of its functional configuration(Wilson and Nock, 2001). Clearly, this is undesirableif protein ligands are sought to functional versionsof targets. A solution to this is to create one, or alimited number of tighter interactions between sup-port and target – for example by using biotinylatingtargets (Hoogenboom et al., 1998). More innovativescreening methods have also been employed includ-ing panning against whole fixed or living cells, tissuesections or even within living animals (Watters et al.,1997; Johns et al., 2000). Screens may also be de-signed such that specific complexes can be selectedfor. One example of this is infectivity screening basedon phage bearing truncated, non-infective fusion coatproteins. Infectivity is restored only if a complex isformed with a binding partner that has the capacityto restore infective functionality to the truncated coatprotein.

Washing and elution

The basic purpose of washing is to remove non-binding phage from the selection process so thatbinding phage are selectively enriched. However, this

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step is worth some consideration because a balanceis required between specificity and avidity of selectedclones. Most phage display libraries of whatever sortare likely to contain clones with a spectrum of aviditiesfor any particular target. Some may be strong binderswith low specificities, others the reverse. If washingis too stringent then highly specific, but weak bindersmay be lost. If washing is not stringent enough thenpopulations of selected clones may be dominated bystrong binders with low specificity. In practice this bal-ance is achieved by adjusting washing times, detergentconcentrations and using regimes in which washingstringencies are progressively increased. A number oftreatments can be used to elute bound phage from tar-gets. Dramatically lowering or increasing pH is oftenemployed, or reducing agents may be used to disruptdisulphide-based links between supports and targets.A more subtle approach using enzymatic cleavage canbe used where there are concerns about the effects onphage integrity of harsh elution conditions. Enzymecleavage sites can be incorporated into the fusion pro-tein, for example a trypsin cleavage site can be insertedbetween M13 pIII and the displayed fusion protein(Rondot et al., 2000).

Re-infection into host cells

It is usually assumed that following elution of boundphage, it is always essential to then amplify the re-covered phage population before the next round ofbio-panning, and indeed virtually all protocols includethis step. However, this dogma may be worth carefulexamination since some reports indicate that directlyusing eluted phage without amplification may reducebackground problems and help reduce the number ofnon-specific phage that are inevitably carried throughthe panning process. The rational is that during am-plification, phage with inferior avidities for the targetbut better growth characteristics may be preferentiallyamplified. This has some important practical implica-tions. The in vivo amplification steps are the most timeconsuming part of phage display library screening andif they could be avoided the time required for eachscreen would therefore be greatly reduced. Moreover,without the in vivo steps it is much easier to envisagehow the whole screening process could eventually becompletely automated (Hoogenboom et al., 1998).

What can be expressed at the surface of phage?

The first incarnation of phage display involved the en-richment of just one expressed protein against a wildtype phage population (Smith, 1985). In the inter-vening seventeen years the scale and scope of phagedisplay has vastly increased. Natural and syntheticpeptides, proteins and protein domains and syntheticantibodies are now all routinely displayed on phage(Winter, 1998a; Winter et al., 1994; Kay and Hoess,1996).

Phage display of peptides and proteins

The starting point of much peptide phage display workis the generation of random combinatorial librariesthat provide a pool of variants from which peptidescan be isolated by affinity selection. The peptides dis-played in these libraries typically range in length from5 to 20 amino acids and in some cases the conforma-tional flexibility of displayed peptides is constrainedby cyclisation. This is likely to afford some protectionagainst proteolysis and may yield peptides with higheraffinities. Cyclisation can be achieved by an amidebond between the N-alpha group and the side chainof the last residue or by a disulphide bridge betweencysteine residues positioned at the N- and C-termini.A number of peptide libraries are freely available fromthe Laboratory of George Smith, University of Mis-souri (Table 1). Random peptide libraries are a sourceof binding partners for a wide range of targets and insome cases the objective of phage display is to sim-ply use isolated peptides directly as molecular probesor agonists. However, peptides may also be isolatedwith sequence homology to the natural protein bind-ing partners of targets and such ‘convergent evolution’studies are a powerful application of peptide phagedisplay (Kay and Hoess, 1996).

Convergent evolution

The theory of convergent evolution of peptides is thatby affinity selection, peptides can be isolated from adiverse starting pool that interact with a given target.Furthermore, the isolated peptides may have sequencehomology to the natural binding partners of the target.Therefore, if the genome of the organism in ques-tion has been sequenced to a significant extent, thenthe sequences from selected phage displayed peptidescan be used to identify their natural counterparts by

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Figure 4. Convergent evolution The natural protein binding partners of a given target may be identified by isolating phage displayed peptidesthat bind to the target and comparing them to a database of native sequences. (a) A population of variant nucleotide sequences are package intophage to generate a phage library displaying variant peptides (b). (c) Phage are screened against a target and binding peptides isolated (d). (e) Adatabase for the organism in question is then searched for sequences with homology to the sequences encoding peptides carried by bindingphage. Genes containing sequences with such homology may then be considered as candidates that encode the natural binding partner of thetarget.

homology comparison (Kay et al., 2000) (Figure 4).One obvious danger is that such screens will isolatenot peptides with homologous sequences, but ‘mimo-topes’ - peptides that bind just as tightly to the targetas the natural binding partner, but have no resem-blance to it at the sequence level. However, variousscreening strategies have been developed to minimisethis outcome (Rodi and Makowski, 1999) and conver-gent evolution has proved to be a powerful strategyfor unravelling protein interaction networks. Never-theless, it perhaps seems surprising that short peptidescan mimic so closely the interactive characteristics ofoften much larger and more complex natural counter-parts. The explanation lies in the fact that for manyproteins only a small subset of residues account formost of the change in free energy that mediates bind-ing. For example human growth hormone consists of217 resides but eight of these account for 85% of thebinding energy (Rodi and Makowski, 1999).

Directed evolution

Once a population of peptide or protein ligands hasbeen isolated, further layers of modification and selec-tion can be applied in order to enhance or manipulatebinding properties or affinities (Figure 5). The strategyof directing a population of peptides or proteins to-wards specific properties by creating random sequencevariation is known as directed evolution. In contrast torational approaches for manipulating the properties ofproteins, directed evolution has the advantage that pro-

teins can be manipulated without the need for a priorknowledge of molecular structure, or of the details ofmolecular action. Using directed evolution it has beenpossible to identify stronger binding ligands to recep-tors, and to produce novel enzyme inhibitors and DNAbinding proteins (Lowman and Wells, 1993; Dennisand Lazarus, 1994; Choo et al., 1994). The productsof convergent evolution experiments can be a fruitfulsource of variants upon which further diversity canbe imposed. Using the sequences encoding isolatedpeptides as a starting point, a second combinatoriallibrary may be generated that is varied around selectedsequences. The starting point for directed evolutioncan also be a protein of which the function is alreadyknown and characterised. A number of strategies areemployed to introduce limited variation, including er-ror prone PCR, the amplification of phage populationsin mutator strains of host bacteria and DNA and fam-ily shuffling. A recent example of this approach isthe creation of variant forms of phytocystatin proteaseinhibitors (McPherson and Harrison, 2001). It hasbeen demonstrated that protease inhibitors expressedin plants under the control of appropriate promoterscan confer resistance to plant parasitic nematodes. Ofthe more than 60 phytocystatin sequences now known,eleven were chosen and subjected to family shuffling.The library of variants is now being screened with theintention of isolating phytocystatins with more potentinhibitory characteristics.

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Figure 5. Directed evolution Phage display is a powerful tool for molecular evolution. A phage library displaying peptides or proteins isscreened against a target and the binding properties of selected peptides or proteins are assessed by an appropriate assay. The nucleotidesequences encoding the selected peptides or proteins are then altered, for example by error prone PCR or DNA shuffling to create a newpopulation of variant nucleotides. The phage display, bio-panning and analysis steps are then repeated in the hope of finding peptides orproteins with altered or improved binding properties.

Directed evolution is also an important tool for themanipulation of enzyme characteristics and displayedvariants of a given enzyme may be rapidly screened foraltered properties. This approach is illustrated by theselection of lipase variants from an M13 phage library(Danielsen et al., 2001). Nine amino acids close tothe active site of lipase from Thermomyces lanuginosawere targeted for randomisation by cassette mutage-nesis and three rounds of selection were performedagainst a biotinylated inhibitor. Analysis of 84 activeclones did not identify enzymes with greater activitythan wild type but sequencing of the diversified regiondid provide insights into the mode of action of thisenzyme.

An adaptation of phage display – substrate phage,may also be used for the analysis of protease sub-strate specificities. With this technique, the displayedmoieties consist of peptides that are potential proteasesubstrates. The peptides are sandwiched between aphage coat protein and a tag (such as c-myc) thatserves to anchor the phage particle to a support. Whenexposed to a particular protease, only phage displayinga cleavable peptide are released into solution, whilstphage displaying non-cleavable peptides remain im-mobilised to the support. The released phage maybe retrieved and amplified and the sequencing of the

inserts from recovered phage provides informationabout the substrate specificity of the protease used(Matthews 1996).

Phage display of antibodies

The worth of antibodies in plant research is well es-tablished (Willats et al., 2002b). In addition to theiruses for detection and isolation of cellular compo-nents, antibodies have the unique capacity when usedas immunocytochemical probes to provide contextualinformation at the sub-cellular level about defined epi-tope structures (Willats et al., 1999; Willats et al.2000). Although the number of antibodies directedagainst plant epitopes has grown steadily over recentdecades they still cover only a minute fraction of themolecular structures involved in plant growth and de-velopment and this shortfall is reflected by gaps inour understanding. Antibody phage display not onlygreatly extends our capacity to generate antibodies butalso extends their potential applications for the directfunctional analysis of epitopes. A further major advan-tage of antibody production by phage display is thatin many cases the whole process can be performed invitro, thereby negating the requirement for target anti-gens to be immunogenic. The range of feasible target

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antigens is therefore extended considerably because amajor limitation for hybridoma antibody productionis the lack of immunogenicity of potential targets.This is particularly true of glycan epitopes and is afactor that has seriously hampered antibody produc-tion against carbohydrate plant cell wall components(Willats et al., 2000). The amount of target antigen re-quired for antibody phage display is much less than istypically required for hybridoma antibody production(micrograms compared to milligrams) and the time re-quired to generate monoclonal antibodies is also muchreduced (a few weeks compared to several months).Because immunisation is by-passed (if single-pot li-braries are used) the ethical and financial burdens ofanimal use are also avoided and phage display anti-body production is relatively simple and cheap andrequires no special facilities.

The principle of antibody phage display

Both conventional hybridoma and phage display an-tibody production exploit the vast diversity of themammalian antibody repertoire. The fundamental dif-ference is that with hybridoma antibody productionthis diversity is harnessed by the immortilisation ofantibody producing B-cells, while with phage displayit is the genes that encode antibody variable regions(V-genes) that that are immortilised. (Winter et al.,1994; Marks et al., 1991; Clackson et al., 1991;Hoogenboom et al., 1998).

The principles of antibody phage display are iden-tical to those discussed above in relation to the displayof peptides and proteins. However, with antibodyphage display the sequences encoding the displayedproteins are derived from genes encoding the key el-ements of natural antibodies that determine binding.The procedures of affinity selection and screening fordesired specificity are essentially the same as those de-scribed for peptide libraries and to some extent mimicthe processes of clonal selection and expansion in themammalian immune system that underpin natural anti-body production. Using directed evolution the bindingproperties of phage antibodies can be further biasedtowards a given target - a process analogous to affinitymaturation in mammals.

Antibody phage display libraries

The mammalian V-genes that encode antibody vari-able domains provide the raw materials for phageantibody library construction. Libraries essentially fallinto two categories depending on whether these genes

are derived from non-immunised animals or animalsimmunised with the target antigen – single-pot andpost immunisation libraries respectively (Figure 6a–c).

Post-immunisation libraries. In the construction ofpost-immunisation libraries, IgG sequences are gen-erally derived from the spleen B-cells of immunisedanimals (Figure 6a). The repertoires of isolated V-genes are manipulated and packaged into phage li-brary vectors. The rational is that some selection andaffinity maturation of sequences with specificity forthe antigen will have already occurred in vivo. Post-immunisation libraries may therefore be pre-biasedtowards containing antibody fragments with desirablespecificities and affinities (Hoogenboom et al., 1998).This approach has been used to create a number ofvaluable antibody probes against plant cell compo-nents. Williams et al. (1996) generated a Fab fragmentwith specificity for the rhamnogalacturonan II (RG II)domain of the pectic cell wall matrix (Williams et al.,1996) while Shinohara et al. (2000) isolated a scFvfrom an post-immunisation library with specificityfor the hemicellulosic fraction of Zinnia cell walls.Although high affinity antibodies can be producedusing post-immunisation phage display libraries thisapproach has several drawbacks. Most serious is thenecessity to construct a new library for every antigenso that the logistical, financial and ethical burdens ofanimal use associated with hybridoma antibody pro-duction are not avoided. Moreover, because of the invivo stage this approach requires target antigens to beimmunogenic.

Single-pot libraries. Because of the limitations out-lined above, a major goal over the past decade hasbeen to create highly diverse, universal, antigen-unbiased libraries from which antibody fragmentswith specificities for a wide range of targets can beisolated. Such single-pot libraries completely avoidimmunisation, library construction for all but the firstuser, and any immunogenic requirement. For thesereasons single-pot libraries have been widely adoptedand used to generate highly specific antibodies to awide range of targets.

Two types of single-pot library have been devel-oped – naïve and synthetic (Figure 6b and c). In un-immunised animals the primary, unselected antibodyrepertoire is dominated by IgMs with a specificity fora variety for antigens. For naïve library construction,v-gene sequences that have undergone some in vivo

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Figure 6. An overview of phage display antibody library construc-tion (a) Post-immunisation libraries are constructed using antibodygene sequences derived from animals that have been immunisedwith the target of interest. This approach capitalises on in vivo an-tibody production processes, such as affinity maturation and mayproduce high affinity antibodies. However, a significant disadvan-tage is that a new library must be constructed for each antigen.In contrast, single-pot libraries (b) and (c) may be used as auniversal resource for the selection of antibodies against a widerange of targets. Naïve libraries (b) are constructed using V-genessequences that have undergone some natural rearrangement, for ex-ample sequences derived from IgM mRNA. Synthetic libraries (c)are constructed ‘from scratch’ using un-arranged germline V-genesequences. (d) Antibody gene sequences are arranged and pack-aged to produce expressed antibody fragments in various formatsincluding single chain antibody fragments (scFvs), Fab fragmentsand disulphide stabilised scFvs (dsFvs).

rearrangement are derived from the IgM mRNA of anun-immunised animal (Figure 6b). This need not bean invasive process since mRNA can be sourced fromperipheral blood lymphocytes (Marks et al., 1991).

Synthetic libraries are ‘built’ in vitro from un-rearranged antibody gene segments with some crit-ically positioned additional random sequences (Fig-ure 6c). The design of synthetic libraries is basedon knowledge of the key CDR (complementarity de-termining region) sequences that shape the antigencombining site and are therefore critical for bind-ing (Winter, 1998b; Hoogenboom and Winter, 1992).Broadly speaking the success of naïve libraries relieson their sheer size while with synthetic libraries thecontents and overall diversity can be designed andcontrolled. Indeed, synthetic antibody libraries are be-ing constructed that are tailor made for given epitopes(Kirkham, et al., 1999; Winter, 1998b).

Two M13-based libraries produced by the Win-ter group at the MRC Centre for Protein Engineering(U.K.) have been widely used and have yielded highaffinity scFvs to diverse targets. Both the SyntheticscFv Library (#1) and Human Synthetic VH+VLscFv Library libraries have been made available tothe scientific community (Table 1). Using the Syn-thetic scFv Library (#1) we have isolated scFvs withspecificity for both protein and carbohydrate targets.Antibody PAM1 binds specifically to un-esterified ho-mogalacturonan (HG, a component of the cell wallpectic matrix) (Willats et al., 1999) while PAM5 bindsspecifically to the tobacco GATA transcription fac-tor TGAF (unpublished results). Using the HumanSynthetic VH+VL scFv Library we have generateda panel of antibodies with specificity for acetylatedHG. In this case phage display was successfully usedafter a hybridoma-based approach failed to yield anti-acetylated HG antibodies.

Antibody formats

Displayed antibody fragments can be configured ina variety of formats (Figure 6d). In the simplestarrangement, scFvs consist simply of a linear chain ofnatural-antibody derived heavy (VH) and light chain(VL) domains joined by additional flexible linker se-quence. Fragments can also be designed with an engi-neered intermolecular disulphide bond that stabilisesthe VH-VL pair (dsFvs). The display of antibody Fabfragments can be achieved by fusing one chain to theC-terminus of pIII and expressing the other chain un-fused and secreted into the periplasmic space of host

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cells where the two chains then associate (Hoogen-boom et al., 1998). A similar approach can be used toform bivalent and bispecific antibody fragments. Be-cause of the small size of the inserts scFv libraries tendto be more genetically stable then Fab libraries. On theother hand scFvs are prone to dimer- and trimerisa-tion which can hamper selection and characterisationof specificity.

Finally, the tantalising prospect of phage particlesthat are not based on, but mimic antibody bindinghas been raised by a strategy known as landscapephage display. This approach does not rely on anti-body derived sequences at all but involves the displayof thousands of copies of a peptide that cover as muchas 50% of the phage surface (for example by fusion tothe pVIII coat protein of filamentous fd-tet) (Petrenkoand Smith, 2000). The spatial limitations imposed bythe packed phage surface serve to constrain the dis-played peptides into a defined organic surface thatcollectively has the capacity to bind to targets withhigh affinity and specificity. Since each ‘landscape’varies according to the displayed peptide, a high di-versity of specificities may potentially be generated.

Using phage display antibodies

In broad terms, phage display and hybridoma-derivednatural antibodies may be used in the same rangeof applications, for example ELISAs, western blotsand immunocytochemistry. However, phage antibod-ies have some particular limitations and advantages.One limitation of hybridoma antibodies is that bindingis usually most effective at mammalian physiologi-cal conditions which can be a disadvantage for someplant research applications. For example, a poten-tially powerful application of antibodies is to directlydisrupt target antigens in vivo However, for manyhybridoma antibodies the conditions required for bind-ing, or even solubility, are not compatible with plantgrowth. With phage display it is possible to regulatescreening conditions such that antibodies with bindingcapacity under defined conditions are isolated.

Discussed below are two aspects of phage antibodyuse in relation to plant science, their use as molecu-lar probes, and for the in vivo immunomodulation oftargets.

Figure 7. The use of phage display antibodies – whole phage versesantibody fragments Both whole phage particles (a) and isolated anti-body framents (b) may be used as immunological probes in a similarrange of applications as hybridoma antibodies. The use of wholephage particles has the disadvantage that their large size leads topoor resolution. (c) In this example of immunogold labelling oftomato pericarp cell walls, the homogalacturonan epitope recog-nised by the M13-based antibody PAM1phage is restricted to themiddle lamella between adjacent cells. Arrowheads indicate theapproximate extent of the middle lamella. However, PAM1phageparticles are in the order of 1 µm long and covered in the pVIIIcoat protein that is the epitope recognised by the gold-conjugatedsecondary antibody. The resulting labelling therefore extends farbeyond the position of the epitope since the whole length of phageparticles is visualised (indicated by arrows). (d) Similarly, whenPAM1phage are used for immunofluorescent microscopy their largesize results in poor resolution ‘fuzzy’ images, as shown by this im-age of PAM1phage binding to cells of tobacco stem parenchyma.(e) In contrast, if the relatively small (∼ 30 KD) free PAM1scFvsare used as probes resolution is greatly increased, as shown by thisimage of PAM1scFv binding to cells of tobacco stem parenchymasimilar to those shown in (d). PAM1scFv was detected via anN-terminal poly-histidine tag. Thanks to Dr Carolina Orfila (KVL,København) for the image shown in (c).

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Figure 8. Antibody and antigen microarrays Microarrayed antibodies can be used to detect antigens, while microarrayed antigens can be usedto detect antibodies. (a) Antibody microarrays are analogous to DNA microarrays in that they can be used to determine, in parallel, the relativeabundance in complex mixtures of molecules isolated from two or more sources, for example mutant and wild type tissues. Differentially dyelabelled extracted molecules are captured by microarrayed natural antibodies (i), antibody fragments (ii) or a mixture of antibody types (iii).The contribution of each dye to the total signal collected from a given spot is a measure of the relative abundance in the samples tested of themolecule recognised by the antibody immobilised on that spot. (b) Antigen microarrays arrays may be used to assess the binding of antibodiesto immobilised antigens, for example to determine antibody specificity. Depending of the microarray platform used a wide range of antigensmay be immobilised including proteins (i) carbohydrates (ii), or glycoproteins (iii). Binding to microarrayed antigens may be detected usingfluorophore conjugated secondary antibodies (iv and v) or by GFP tagging or dye labelling of antibodies (vi and vii). (c) We have used antigenmicroarrays to assess the binding of phage display antibodies with specificity for acetylated homogalacturonan (AceHG) domains of cell wallpectic polysaccharides. Ten replicates (I-X) of ten different (1–10) AceHG samples were microarrayed onto polystyrene MaxiSorpTM treatedslides (Nunc, Denmark). The differential binding of three (i–iii) different phage display monoclonal antibodies is shown. Antibody binding wasdetected using anti-M13pVIII /FITC secondary antibodies.

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Phage antibodies as immunocytochemical probes

Phage antibody binding can be detected by the use ofsecondary antibodies with specificity for phage coatproteins. For example PAM1phage (Figure 7a) can bedetected using secondary antibodies with specificityfor the M13 pVIII coat protein. The use of anti-pVIII secondary antibodies effectively amplifies scFvbinding because of the approximately 2,700 copiesof pVIII that coat the M13 phage particles (see Fig-ure 3d). However, the large size of M13 is a disad-vantage for immunocytochemical localisation studiesbecause of the diffuse signal resulting from secondaryantibody binding to the multiple copies of pVIII dis-tributed along the phage particle (Figure 7c and d).In order to overcome this it is necessary to use solu-ble (non-phage bound) scFvs. These can be producedby amplifying PAM1 in a non-suppressing host al-though we found that this approach was inefficient.Instead, we cloned the PAM1 coding sequence fromthe phagemid (pHEN1, Nissim et al., 1994) into abacterial expression vector and at the same time addeda poly-histidine tag to the scFv. The soluble form ofPAM1 (known as PAM1scfv, Figure 7b) can be readilyisolated to high purity using a nickel resin column.PAM1phage and PAM1scfv have identical specificitiesalthough the detection limit of PAM1scfv is less thanthat of PAM1phage because the poly-histidine tag pro-vides more limited binding possibilities for each sec-ondary antibody compared to the numerous pVIII coatproteins. However, when used for immunocytochem-ical labelling the small size of PAM1scfv providesmuch superior resolution compared to PAM1phage(Figure 7e).

Recently, the immunocytochemical applications ofphage display antibodies have been extended by theproduction of scFv/GFP fusions. Functional analysishas established that in many cases such fusions can bemade in which both the scFv and GFP moieties retaintheir original activities (Morino et al., 2001; Caseyet al., 2000). Apart from providing convenient probesfor quick and simple one-step immunocytochemistry,the exciting possibility is also raised of being ableto express scFv/GFP fusions in planta for real timeintracellular labelling of given epitopes during devel-opmental processes. Moreover, panels of co-expressedfluophor tagged scFvs could also be used for the invivo analysis of molecular interaction using fluores-cence resonance energy transfer (FRET) (Truong andIkura, 2001; Gadella et al., 1999).

Immunomodulation

Several powerful approaches are available for the dis-ruption of plant processes and molecules at the genelevel. However, if the genetic pathways controlling aparticular process or molecule are not characterised analternative strategy is to directly disrupt gene prod-ucts in order to elucidate their functions. One suchdirect approach is immunomodulation - the disruptionof antigen function by the action of antibody binding(Smith and Glick, 2000). This can be achieved eitherby micro-injection of antibodies into cells, incorpora-tion of antibodies into plant or plant cell growth media,or by the expression of antibodies in plants. This lastapproach has the most practical potential for func-tional analysis of a wide range of intracellular antigensin vivo.

One problem associated with the expression ofwhole antibodies or Fab fragments in plants is thatthe intracellular environment is not conducive to cor-rect antibody assembly. In this regard scFvs, withtheir relatively undemanding folding requirements, areparticularly well suited for this role and have been suc-cessfully used to immunomodulate a variety of plantantigens (De Jaeger et al., 2000). Immunomodula-tion of the activity of the plant hormones abscisicacid (Strauß et al., 2001) and gibberellin (Shimadaet al., 1999) and the receptor protein phytochrome(Owen et al., 1992) has been demonstrated. Antibod-ies have been targeted to the cytosol, the endoplasmicreticulum, and apoplast but in theory any cellularcompartment can be targeted. However, immunomod-ulation using scFvs is not always straightforward andmany expressed scFvs are unstable. One solution is todevelop more stable scFv scaffolds, another is to buildantibodies free of disulphide bonds (Hoogenboomet al., 1998). Another interesting approach exploitsthe unusual antibodies of the Camelidae (Hamers-Casterman et al., 1993). In addition to four chainantibodies, the Camelidae produce antibodies con-taining only heavy chains (Hamers-Casterman et al.,1993). Simple single-domain fragments (VHH) de-rived from these heavy chain antibodies may be avaluable resource for immunomodulation (De Jaegeret al., 2000).

Microarrays for characterising and using phagedisplay antibodies

The development of DNA microarrays has been oneof the most significant bio-technological advancesin recent years and is set to revolutionise the high

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throughput analysis of gene expression (Lander, 1999;Debouck and Goodfellow, 1999). Microarray tech-nology is increasingly applied to the analysis of pro-tein interactions and the analysis of antibody binding(Tomlinson and Holt, 2001; Kodadek, 2001). Arraysof antibodies (antibody arrays) can be used to detectantigens, whilst arrays of antigens (antigen arrays) canbe used to detect antibodies (Haab et al., 2001).

Antibody arrays (Figure 8a) can be used for pro-teome profiling. The rational is to isolate ligandsfrom complex mixtures on the basis of their bindingto immobilised antibodies. A typical experiment in-volves the isolation of mixtures of proteins from say,experimental and control cells or tissue. Each pro-tein mixture is then bulk labelled with distinguishablemarkers (such as Cy3 and Cy5) and exposed to theimmobilised antibodies. The contribution of each dyeto the total signal collected from a given spot is ameasure of the relative abundance of the moleculerecognised by the antibody immobilised on that spot.This parallel analysis is analogous to typical DNA mi-croarray experiments. Antigen arrays (Figure 8b) areessentially very high throughput versions of ELISAor dot-blot assays in which the binding capacity ofa particular protein ligand or antibody is assessedby its binding to a series of microarrayed potentialbinding partners. Binding can be detected directly iffluorophore-coupled proteins or antibodies are used orby using fluorescently labelled secondary antibodies(Figure 8b).

In our experience the time limiting step in phageantibody production is the detailed analysis of eachmonoclonal phage population. Conventional assays,such as ELISAs and immuno-dot-assays have the dis-advantages that only a relatively small number of sam-ples can be tested simultaneously, and large amountsof antibody are required for each assay. Using pro-tein antigen microarrays, many thousands of potentialbinding partners can be assessed simultaneously us-ing a very small (< 100 µl) amount of phage solutionamplified simultaneously in microtitre plates. Slidesurfaces coated with polylysine or super-aldehydes areavailable for the microarray deposition of proteins butequivalent surfaces are not available for the prepara-tion of carbohydrate microarrays. To address this, anovel polymer microarray slide has recently been de-veloped. The slide surface has capacity to immobilisestructurally and chemically diverse glycans withoutany derivatisation of the slide surface or the need tocreate reactive groups on the glycans prior to immo-bilisation (Willats et al., 2002a). The slides are made

of polystyrene and have a surface modification knownas MaxiSorpTM (NUNC A/S, Denmark) – a surfacethat has been widely used in a microtitre plate for-mat for ELISAs for many years. Using these slideswe have generated carbohydrate microarrays and usedthem to characterise phage display antibodies withspecificities for plant cell wall pectic polysaccharides(Figure 8c).

Conclusions

Phage display is a multi-purpose tool for amongstother things, molecular evolution, analysis of pro-tein/ligand interactions and the generation of antibod-ies. However, the relative scarcity of plant-specificexamples of some applications of phage display re-flects the fact that this technology has much still tooffer plant research. As the post-genomic era pro-gresses the emphasis of research is likely to focusincreasingly on making sense of the biological con-texts of gene products. In this respect many of theapplications of phage display outlined here will bevaluable tools.

Acknowledgements

Thanks to Iain Manfield, Sue Marcus, Lesley McCart-ney, Jürgen Denecke, Carolina Orfila, Jørn DalgaardMikkelsen, NUNC A/S and Eva Maria Klein.

References

Casey, J.L., Coley, A.M., Tilley, L.M. and Foley, M. 2000. Greenfluorescent antibodies: novel in vitro tools. Protein Engin. 13(6):445–452.

Castillo, J., Goodson, B. and Winter, J. 2001. T7 displayed peptidesas targets for selecting peptide specific scFvs from M13 scFvdisplay libraries. J. Immunol. Meth. 257: 117–122.

Choo, Y., Sánchez-Garcia, I. and Klug, A. 1994. In vivo repres-sion by a site-specific DNA-binding protein designed against anoncogenic sequence. Nature 372: 642–645.

Clackson, T., Hoogenboom, H.R., Griffiths, A.D. and Winter, G.1991. Making antibody fragments using phage display libraries.Nature 352: 624–628.

Danielsen, S., Eklund, M., Deussen, H-J., Graslund, T., Nygren,P-Å. and Borchert, T.V. 2001. In vitro selection of enzymaticallyactive lipase variants from phage libraries using a mechanism-based inhibitor. Gene 272: 267–274.

Danner, S. and Belasco, J.G. 2001. T7 phage display: a novel ge-netic selection system for cloning RNA-binding proteins fromcDNA libraries. Proc. Nat. Acad. Sci. USA 98(23): 12954–12959.

853

Debouck, C. and Goodfellow, P.N. 1999. DNA microarrays in drugdiscovery and development. Nature Genetics (supplement) 21:48–50.

De Jaeger, C., De Wilde, C., Eeckhout, D., Fiers, E. and Depicker,A. 2000. The plantibody approach: expression of antibody genesin plants to modulate plant metabolism or to obtain pathogenresistance. Plant Mol. Biol. 43: 419–428.

Dennis, M.S. and Lazarus, R.A. 1994. Kunitz domain inhibitors oftissue-factor VIIa. I. Potent inhibitors selected from libraries byphage display. J. Biol. Chem. 269: 22129–22136.

Drees, B.L. 1999. Progress and variations in two-hybrid and threehybrid technologies. Curr. Opin. Chem. Biol. 3: 64–70.

Gadella, T.W.J., van der Krogt, G. N. M. and Bisseling, T. 1999.GFP-based FRET microscopy in living plant cells. Trends PlantSci. 4(7): 287–291.

Griffiths, A.D., Malmqvist, M., Marks, J.D., Bye, J.M., Embleton,M.J., McCafferty, J., Baier, M., Holliger, K.P., Gorick, B.D.,Hughes-Jones, N.C., Hoogenboom, H.R. and Winter, G. 1993.Human anti-self antibodies with high specificity from phagedisplay libraries. EMBO J. 12(2): 725–734.

Haab, B.B., Dunham, M.J. and Brown, P.O. 2001. Protein microar-rays for highly parallel detection and quantification of specificproteins and antibodies in complex solutions. Genome Biol. 2(2):1004.1–1004.13.

Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robin-son, G., Memaers, C., Bajyana Songa, E., Bendahman, N. andHamers, R. 1993. Naturally occurring antibodies devoid of lightchains. Nature 363: 446–448.

Hoogenboom, H.R. 1997. Designing and optimising library selec-tion strategies for generating high-affinity antibodies. TIBTECH15: 62–70.

Hoogenboom, H.R., de Bruïne A.P., Hufton, S.E., Hoet, R.M.,Arends, J-W. and Roovers, R.C. 1998. Antibody phage displayand its applications. Immunotechnology 4: 1–20.

Hoogenboom, H.R. and Winter, G. 1992. By-passing immunisation.Human antibodies from synthetic repertoires of germline VHsegments rearranged in vitro. J. Mol. Biol. 227: 381–388.

Johns, M., George, A.J.T. and Ritter, M.A. 2000. In vivo selectionof scFv from phage display libraries. J. Immunol. Meth. 239:137–151.

Kay, B.K. and Hoess, R.H. 1996. Principles and applicationsof phage display. In: B.K. Kay, J. Winter and J. McCafferty(eds.) Phage display of peptides and proteins, Academic Press,pp. 21–34.

Kay, B.K., Kasanov, J., Knight, S. and Kurakin, A. 2000. Con-vergent evolution with combinatorial peptides. FEBS Lett. 480:55–62.

Kirkham, P.M., Neri, D. and Winter, G. 1999. Towards the design ofan antibody that recognises a given protein epitope. J. Mol. Biol.285: 909–915.

Kodadek, T. 2001. Protein microarrays: Prospects and problems.Chem. Biol. 8: 105–115.

Lander, E.S. 1999. Array of hope. Nature Genetics (supplement) 21:3–4.

Lowman, H.B. and Wells, J.A. 1993. Affinity maturation of humangrowth hormone by monovalent phage display. J. Mol. Biol. 234:564–578.

Morino, K., Katsumi, H., Akahori, Y., Iba, Y., Shinohara, M.,Ukai, Y., Kohara, Y. and Kurosawa, Y. 2001. Antibody fusionswith fluorescent proteins: a versatile reagent for profiling proteinexpression. J. Immunol. Meth. 257: 175–184.

Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J.,Griffiths, A.D. and Winter, G. 1991. By-passing immunisation.

Human antibodies from V-gene libraries displayed on phage. J.Mol. Biol. 222: 581–597.

Matthews, D.J. 1996. Substrate phage. In: B.K. Kay, J. Winter andJ. McCafferty (eds.), Phage display of peptides and proteins,Academic Press, pp. 255–259.

Mendelsohn, A.R. and Brent, R. 1999. Protein interaction methods– towards an endgame. Science 18(284): 1948–1950.

McCafferty, J. 1996. Phage display: factors affecting panning effi-ciency. In: B.K. Kay, J. Winter and J. McCafferty (eds.), Phagedisplay of peptides and proteins, Academic Press, pp. 261–276.

McCafferty, J. and Johnson, K.S. 1996. Construction and screen-ing of antibody display libraries. In: B.K. Kay, J. Winter andJ. McCafferty (eds.), Phage display of peptides and proteins,Academic Press, pp. 79–111.

McPherson, M.J. and Harrison, D.J. 2001. Protease inhibitors anddirected evolution: enhancing plant resistance to nematodes. In:A. Berry and S.E. Radford (eds.), From protein folding to newenzymes, Cambridge University Press, Cambridge, U.K.

Nissim, A., Hoogenboom, H.R., Tomlinson, I.A., Flynn, G., Midg-ley, C., Lane, D. and Winter, G. 1994. Antibody fragments froma ‘single-pot’ phage display library as immunological reagents.EMBO J. 13: 692–697.

Owen, M., Gandecha, A., Cockburn, B. and Whitelam, G. 1992.Synthesis of a functional anti-phytochrome single-chainFv pro-tein in transgenic tobacco. Bio/technology 10: 790–794.

Petrenko, V.A. and Smith, G.P. 2000. Phages from landscapelibraries as substitutes for antibodies. Protein Engin. 13(8):589–592.

Rodi, D.J. and Makowski, L. 1999. Phage-display technology– finding a needle in a vast molecular haystack. Curr. Opin.Biotechnol. 10: 87–93.

Rodi, D.J., Makowski, L. and Kay, B.K. 2001. One from columnA and two from column B: the benefits of phage display inmolecular-recognition studies. Curr. Opin. Chem. Biol. 6: 92–96.

Rondot, S., Koch, J., Breitling, F. and Dübel, S. 2001. A helperphage to improve single-chain antibody presentation in phagedisplay. Nature Biotechnol. 19: 75–78.

Sidhu, S.S. 2001. Engineering M13 for phage display. Biomol.Engin. 18: 57–63.

Shimada, N., Suzuki, Y., Nakajima, M., Conrad, U., Murofushi,N. and Yamaguchi, I. 1999. Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco. Biosci.Biotechnol. Biochem. 63: 779–783.

Shinohara, N., Demura, T. and Fukuda, H. 2000. Isolation of a vas-cular cell wall-specific monoclonal antibody recognising a cellpolarity by using a phage display subtraction method. Proc. Nat.Acad. Sci. USA 97(5): 2585–2590.

Smith, G.P. 1985. Filamentous phage fusion: novel expression vec-tors that display cloned antigens on the surface of the viron.Science 228: 1315–1317.

Smith, M.D. and Glick, B.R. 2000. The production of antibodiesin plants: an idea whose time has come? Biotechnol. Adv. 18:85–89.

Sparks, A.B., Adey, N.B., Cwirla, S. and Kay, B.K. 1996. Screen-ing phage-displayed random peptide libraries. In: B.K. Kay, J.Winter and J. McCafferty (eds.), Phage display of peptides andproteins, Academic Press, pp. 227–253.

Strauß M., Kauder, F., Peisker, M., Sonnewald, U., Conrad, U.and Heineke, D. 2001. Expression of an abscisic acid-bindingsingle-chain antibody influences the subcellular distribution ofabscisic acid and leads to developmental changes in transgenicpotato plants. Planta 213: 361–369.

Tomlinson, I.M. and Holt, L.J. 2001. Protein profiling cones of age.Genome Biol. 2(2): 1004.1–1004.3.

854

Truong, K. and Ikura, M. 2001. The use of FRET imagingmicroscopy to detect protein-protein interactions and proteinconformational changes in vivo. Curr. Opin. Struct. Biol. 11:573–578.

Watters, J.M., Telleman, P. and Junghans, R.P. 1997. An optimisedmethod for cell based phage display panning. Immunotechnol-ogy 3: 21–29.

Whaley, S.R., English, D.S., Hu, E.L., Barbara, P.F. and Belcher,A.M. 2000. Selection of peptides with semiconductor bindingspecificity for directed nanocrystal assembly. Nature 405: 665–668.

Willats, W.G.T., Gilmartin, P.M., Mikkelsen, J.D. and Knox,J.P. 1999. Cell wall antibodies without immunisation: genera-tion of and use of de-esterified homogalacturonan block-specificantibodies from a naïve phage display library. Plant J. 18: 57–65.

Willats, W.G.T., Rasmussen, S.E., Kristensen, T., Mikkelsen, J.D.and Knox, J.P. 2002a. Sugar-coated microarrays: a novel slidesurface for the high throughput analysis of glycans. Proteomics2(12): in press.

Willats, W.G.T., Steele-King, C.G. and Knox, J.P. 2002b. Antibodytechniques. In: P. Gilmartin and C. Bowler (eds.), MolecularPlant Biology: a practical approach, Oxford University Press,Oxford, U.K.

Willats, W.G.T., Steele-King, C.G., McCartney, L., Orfila, C.,Marcus, S.E. and Knox, J.P. 2000. Making and using antibodyprobes to study plant cell walls. Plant Physiol. Biochem. 38(1–2):27–36.

Williams, M.N., Freshour, G., Darvill, A.G., Albersheim, P. andHahn, M.G. 1996. An antibody Fab selected from a recombinantphage display library detects deesterified pectic polysacchariderhamnogalacturonan II in plant cells. Plant Cell 8: 673–685.

Wilson, D.S. and Nock, S. 2001. Functional protein microarrays.Curr. Opin. Chem. Biol. 6: 81–85.

Winter, G. 1998a. Synthetic human antibodies and a strategy forprotein engineering. FEBS Lett. 430: 92–94.

Winter, G. 1998b. Making antibody and peptide ligands by reper-toire selection technologies. J. Mol. Recogn. 11: 126–127.

Winter, G., Griffiths, A.D., Hawkins, R.E. and Hoogenboom, H.R.1994. Making antibodies by phage display technology. Annu.Rev. Immunol. 12: 433–455.

Uetz, P. 2001. Two-hybrid arrays. Curr. Opin. Chem. Biol. 6: 57–62.Zucconi, A., Dente, L., Santonico, E., Castagnoli, L. and Cesareni,

G. 2001. Selection of lgands by panning of domain librariesdisplayed on phage lambda reveals new potential partners ofsynaptojanin 1. J. Mol. Biol. 307: 1329–1339.