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High-density Functional Display of Proteins onBacteriophage Lambda

Amita Gupta1, Masanori Onda2, Ira Pastan2, Sankar Adhya2 andVijay K. Chaudhary1*

1Department of BiochemistryUniversity of Delhi SouthCampus, New Delhi 110021India

2Laboratory of MolecularBiology, Center for CancerResearch, 37 Convent Dr., Rm.5106, Bethesda, MD20892-4264, USA

We designed a bacteriophage lambda system to display peptides and pro-teins fused at the C terminus of the head protein gpD of phage lambda.DNA encoding the foreign peptide/protein was first inserted at the 30

end of a DNA segment encoding gpD under the control of the lac promo-ter in a plasmid vector (donor plasmid), which also carried lox Pwt and loxP511 recombination sequences. Cre-expressing cells were transformed withthis plasmid and subsequently infected with a recipient lambda phagethat carried a stuffer DNA segment flanked by lox Pwt and lox P511 sites.Recombination occurred in vivo at the lox sites and Ampr cointegrateswere formed. The cointegrates produced recombinant phage that dis-played foreign protein fused at the C terminus of gpD. The system wasoptimised by cloning DNA encoding different length fragments of HIV-1p24 (amino acid residues 1–72, 1–156 and 1–231) and the display wascompared with that obtained with M13 phage. The display on lambdaphage was at least 100-fold higher than on M13 phage for all the frag-ments with no degradation of displayed products. The high-density dis-play on lambda phage was superior to that on M13 phage and resultedin selective enrichment of epitope-bearing clones from gene-fragmentlibraries. Single-chain antibodies were displayed in functional form onphage lambda, strongly suggesting that correct disulphide bond forma-tion takes place during display.

This lambda phage display system, which avoids direct cloning intolambda DNA and in vitro packaging, achieved cloning efficienciescomparable to those obtained with any plasmid system. The high-densitydisplay of foreign proteins on bacteriophage lambda should be extremelyuseful in studying low-affinity protein–protein interactions moreefficiently compared to the M13 phage-based system.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: phage display; recombination; gpD; gene-fragment library;antibody fragment*Corresponding author

Introduction

Display of peptides and proteins on the surfaceof bacteriophage is a powerful approach to studyprotein–protein interactions.1 – 3 The power of thesystem emanates from the direct physical linkage

between the phenotype and its genotype, so that adesired molecule can be easily selected from amilieu of millions and identified by sequencingthe DNA encapsulated in that phage particle.

Phage display technology, which started withthe identification of peptide epitopes recognisedby monoclonal antibodies4 has grown into anapproach for cloning human antibodies, studyingligand–receptor interactions, elucidating signaltransduction pathways, delineating contact resi-dues in interacting proteins, and isolating peptideinhibitors.2,5,6 Other applications of this technologyinclude the production of gene/genome-fragmentand cDNA libraries7 – 10 displaying virtually every

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: cfu, colony-forming units; DCO,double crossover; HRP, horse radish peroxidase; mAb,monoclonal antibody; PAG, polyacrylamide gel; PE,Pseudomonas exotoxin; pfu, plaque-forming units; SCO,single crossover.

doi:10.1016/j.jmb.2003.09.033 J. Mol. Biol. (2003) 334, 241–254

possible encoded peptide/protein that can be usedfor identifying specific interacting sequences.

The bacteriophage M13 has been the mostwidely used system for display of peptides/pro-teins. Fusion to the minor coat protein, gIIIp, andthe major coat protein, gVIIIp, of M13 is used fordisplay of a range of molecules of different sizesand structures. However, the high-density displayof large protein domains on M13 is inefficient andoften associated with extensive degradation.11

Since M13 morphogenesis occurs in the periplasm,it is possible that the molecules that are secretion-incompetent may not get displayed. M13 is used

primarily as an N-terminal display system butsome variants have been developed for C-terminaldisplay12,13 and construction of cDNA libraries.14

There have been reports in recent years describ-ing N and C-terminal display of peptides/proteinson phage lambda as fusion to the capsid protein“d” (gpD) and tail protein “v” (gpV) of lambda.15–17

These systems have yet to gain wide acceptancefor the following reasons: (i) lambda phage biologyis more complex than that of M13; (ii) the lambdagenome is very large (50 kb); as a result, isolationof viral DNA, insertion of user-defined restric-tion sites, cloning of foreign fragments and then

Figure 1. Diagrammatic represen-tation of different vectors. Onlyrelevant genes and restriction sitesare shown. The maps are not toscale. lacPO, lac promoter-operator;RBS, ribosome-binding site; D, seg-ment encoding amino acid residues1–109 of gpD of lambda; Stuffer, a30 nucleotide long sequence;c-myc, decapeptide recognised bymonoclonal antibody, 9E10; fori,origin of replication of filamentousphage f; Ampr, b-lactamase gene;Ori, ColE1 origin of replication; loxPwt, wild-type lox site; lox P511, loxsite with mutation 511. A, Donorplasmid, pVCDcDL1 with cloningsites NheI and MluI. Details ofregions marked as a and b bydouble-headed arrows are shownin Aa and Ab, respectively. Aminoacids are shown in single-lettercode below the nucleotidesequence. Restriction enzyme sitesare shown above the nucleotidesequence. p , translation stop. L7and L15 are oligonucleotide primersused for PCR-based analysisof recombinants. Aa, Sequenceshowing the ribosome-binding siteand the initiation codon inpVCDcDL1. Ab, Sequence showingthe end of gpD coding region,collagenase site, stuffer fragmentand c-myc in pVCDcDL1.B, Recipient phage vector, lDL1.Only some of the lambda genes areshown. Dam, D gene of lambdawith amber mutation. The uniqueXbaI site in the lambda genomeused for cloning is shown. lacZa,DNA cassette comprised of lacPO,RBS and the first 58 codons of lacZ;L1 and L4 are oligonucleotideprimers used for PCR-basedanalysis of cointegrates. C, Donorplasmid pVCDcDL3 is similar topVCDcDL1 but contains betweenthe NheI and MluI sites a lacZcassette comprised of lacPO, RBS

and the first 148 codons of lacZ flanked by SmaI/SrfI restriction enzyme sites. Blunt-ended DNA fragments can becloned into SmaI/SrfI-cut vector and recombinants produce white colonies on X-gal plates. T, universal translation stop.

242 Functional Display of Proteins on Lambda

packaging of the ligated product in vitro to makelambda particles are difficult and the library sizesachieved are smaller than those obtained withphagemid-based M13 vectors;18 and (iii) intracellu-lar assembly of phage may not allow disulphidebond formation in the molecule to be displayed.

Here, we describe a lambda phage displaysystem that allows simplified, high-efficiencycloning in lambda DNA and high-density displayof peptides/proteins fused to gpD of lambda.We compare the display of different molecules onlambda phage and M13 phage and demonstratethe advantage of high-density display on lambdain biopanning for epitope mapping using gene-fragment libraries. We also show that the lambdaphage system is able to display proteins withmultiple disulphide bonds in functional form andin large numbers.

Results

Cloning into lambda display vector byin vivo recombination

The cloning strategy is based on first insertingDNA encoding peptide-protein into a high copydonor plasmid vector, pVCDcDL1 (Figure 1A),and then transferring this genetic information intorecipient lambda genome, lDL1 (Figure 1B), bythe high-efficiency lox-Cre recombination systemin vivo (Figure 2).

pVCDcDL1 contained a sequence encoding gpDof l (Figure 1Aa), followed by a GGSG spacer(s), acollagenase site, NheI site, a stuffer segment, MluIsite and a c-myc tag (Figure 1Ab), under thecontrol of the lac promoter (lacPO). Cloning ofDNA sequences as NheI–MluI inserts in place ofthe stuffer allowed formation of a D fusion proteinwith collagenase site between D and the foreignprotein and c-myc tag at the C terminus. Thevector also contained the M13 phage origin ofreplication ( fori), flanked by lox Pwt and lox P511

recombination sequences. lDL1, the recipientlambda vector, contains a lacZa fragment flankedby lox Pwt and lox P511 recombination sequences atthe unique XbaI site present in the lambda gen-ome. The lox sequences in the donor plasmid arein the reverse orientation to that in the recipientlambda genome (Figure 1B). When Escherichia coliexpressing Cre recombinase (Creþ host) weretransformed with the donor plasmid and theninfected with lDL1, recombination occurred at thecompatible lox sites in the two vectors, resulting inintegration of the plasmid DNA into the lambdaDNA (Figure 2). Note that Cre-mediated recombi-nation occurs between two lox Pwt sites or lox P511

sites, and not between lox Pwt and lox P511 sites.19

Hence, plasmid and lambda DNA crossing overoccurred only in trans and resulted in formationof a cointegrate. Also, due to opposite orientationof the lox sites in the plasmid and lambda, therecombination led to integration of the entireplasmid DNA into the lambda DNA. The first

Figure 2. The process of recombination. lox sites shown in black are of the recipient lambda phage vector. Cre, Crerecombinase; SCO, single crossover cointegrate; DCO, double crossover cointegrate. Filled arrows indicate the direc-tion of transcription from the promoter of b-lactamase, lacZa and D gene. L1 and L4 are oligonucleotides used forPCR-based analysis of cointegrate. Only one of the possible recombination pathways is shown (first crossover at loxPwt followed by second crossover at lox P511). The other pathway (first crossover at lox P511 followed by secondcrossover at lox Pwt) will yield the same product.

Functional Display of Proteins on Lambda 243

crossover event (intermolecular) resulted in theformation of single crossover (SCO) cointegratethat contained the complete donor plasmid inte-grated in the lambda genome. A second crossoverevent (intramolecular) at the other pair of com-patible lox sites resulted in the formation of adouble crossover (DCO) cointegrate and excisionof the lacZa fragment and fori sequence (Figure 2).Thus, the DNA encoding the foreign peptide/protein fused to gpD for display on lambda phagesurface becomes part of the lambda genome. Thelambda also acquires the b-lactamase selectionmarker of the plasmid.

Based upon this strategy, BM25.8 (Creþ host) andTG1 (Cre2 host) were transformed with the donorplasmid, pVCDcDL1, and then infected withrecipient lambda phage, lDL1. The cultures weregrown in ampicillin-containing medium until com-plete cell lysis. The cell-free lysate obtained after therecombination event was used to infect Cre2 cellsand plated to determine plaque-forming units(pfu) and colony-forming units (cfu) (on ampi-cillin-containing medium). As shown in Table 1,the number of pfu was the same in the lysateobtained from Creþ and Cre2 hosts, indicatingsimilar amounts of phage production in bothhosts. However, only the lysate from Creþ hostwas able to transduce Ampr colonies in E. coli.This indicates that the plasmid integrated intolambda DNA only in the presence of Cre pro-tein and conferred ampicillin resistance to cellsharbouring this lambda cointegrate as extrachromosomal lysogen driven by the plasmid repli-con. The lysate obtained from Creþ host containedthree phage species: parental recipient lambda,SCO cointegrate (lDcDL1: SCO) and DCO cointe-grate (lDcDL1: DCO). Plating on ampicillin-containing medium selected for cointegrates andeliminated parental phage. To check for thepresence of plasmid sequence in lambda genome,the Ampr colonies were analysed by PCR usingprimers L1 and L4 (Figure 2) that flank the loxsequences in lambda. Agarose gel electrophoresisof amplified products revealed that all the coloniesanalysed harboured cointegrates and the ratio ofSCO to DCO cointegrates was 1:3 (data notshown). lDcDL1: SCO and lDcDL1: DCO harbour-ing clones were grown in ampicillin-containingmedium wherein there was spontaneous phageproduction leading to cell lysis. The cell-free

lysates obtained were tested for phage titre andpresence of gpD-c-myc protein on the phagesurface. Both SCO and DCO harbouring cellsproduced the same number of phage (Table 2)determined as pfu. To test the stability of phageparticles, the lysates were incubated in EDTA-containing buffer and then re-titrated to determinethe number of viable phages. No difference in pfubefore and after incubation in EDTA was observedfor lysates obtained from SCO and DCO clones,indicating that the phages produced were resistantto EDTA and all 405 copies of gpD (either as gpDor gpD fusion protein) were present on everyphage particle.20 The phage particles were thentested for display of c-myc peptide as gpD fusion.Both types of phage displayed the same amountof c-myc peptide as revealed by equal recovery ofphages (,2% of phages added) following bio-panning in anti-c-myc (mAb 9E10) coated wells(Table 2). This recovery was at least 200-fold higherthan that obtained for lDL1 phage (that does notdisplay gpD-c-myc). Western blot analysis withmAb 9E10 showed that phages purified from lysateof both SCO and DCO clones produced a band of,16 kDa with an intensity corresponding to ,400copies of fusion protein per phage particle (datanot shown; the number of fusion proteins wascalculated by densitometric scanning of the blotusing a purified c-myc-containing protein as con-trol). These experiments established that SCO andDCO phage had similar properties and all ampr

transductants obtained after recombination pro-duced functional phage displaying gpD fusionprotein.

High-density display of peptides and proteinson lambda; a comparison with M13

Display of different size molecules on lambdaphage and a comparison with the M13 phage dis-play system in terms of density and functionalityof displayed peptides and proteins was carried

Table 2. Characterisation of phage produced by SCO andDCO cointegrates

Phage Titre (pfu)a EDTA resistantb Recovery (%)c

lDcDL1: SCO 4 £ 109/ml þ 2.24lDcDL1: DCO 4 £ 109/ml þ 2.22lDL1 4 £ 109/ml þ ,0.01

lDcDL1: SCO, single crossover cointegrates of lDcDL1 dis-playing gpD-c-myc; lDcDL1:DCO, double crossover cointegrateof lDcDL1 displaying gpD-c-myc; lDL1, wild-type recipientphage not displaying c-myc.

a Colonies harbouring SCO and DCO cointegrates of lDcDL1were grown and phage titre in culture lysate determined as pfu.

b Lambda phage were diluted in TM (10 mM Tris, 10 mMMgCl2) or TE (10 mM Tris, 10 mM EDTA) and incubated at37 8C for ten minutes. Serial dilutions of the samples were thentitred on TG1 cells to determine pfu. þ Indicates no differencein the titre of phage in TM and TE diluted samples.

c Bio-panning was done using anti-c-myc mAb, 9E10. Therecovery was calculated as (output titre/input titre) £ 100.

Table 1. Analysis of recombination of recipient vectorlDL1 and donor plasmid pVCDcDL1

Sample pfu/ml cfu/ml

Lysate obtained after recombination inCre2 host

1.5 £ 109 0

Lysate obtained after recombination inCreþ host

1.5 £ 109 2.5 £ 108

Cultures of TG1 (Cre2) and BM25.8 (Creþ) transformed withpVCDcDL1 were infected with lDL1 and the lysate obtainedtitrated as pfu and cfu in TG1.


out using fragments of HIV-1 capsid protein p24.HIV-1 p24 contains two independently foldingdomains.21,22 The first 156 amino acid residues ofp24 constitute the N-terminal domain that interactswith host proteins such as cyclophilin, while resi-dues 157–231 constitute the C-terminal domain,which is responsible for oligomerisation of p24 toform the viral capsid. Three fragments of p24encompassing residues 1–72 (p241), 1–156 (p246,N-terminal domain of p24) and 1–231 (p24, full-length protein) were displayed as C-terminalfusions with gpD on lambda, and as N-terminalfusions with gVIIIp and gIIIp on M13 using phage-mid-based vectors. All the fusion proteinscontained c-myc tag at the C terminus of p24 frag-ment. Phage were prepared for all lambda andM13 clones and purified by polyethylene glycol(PEG) precipitation and ultracentrifugation. Thepurified phages were then tested for binding toanti-p24 mAb in ELISA and the display of fusionprotein on the phage surface was quantified byWestern blot using anti-c-myc mAb 9E10. InELISA, both M13 and lambda phage displayingp24 fragments showed dose-dependent binding tomAb H23, which recognises amino acid residues56–66 of p24 (Figure 3A). p241-displaying phageshowed maximum reactivity followed by p246-

displaying and p24-displaying phage. As seen inFigure 3A, for all the three displayed molecules,lambda phage showed two to three orders ofmagnitude better reactivity compared to corre-sponding M13 phage, indicating higher display ofthe proteins.

The number of fusion protein molecules dis-played per phage particle was quantified byWestern blot analysis using mAb 9E10 (Figure 3B).In the case of lambda phage (Figure 3Ba), anintense band corresponding to the calculated mol-ecular mass was seen for each of the three fusionproteins. The number of fusion protein moleculesdisplayed per phage particle was estimated to be350 copies of gpD-p241-c-myc (22 kDa, Figure 3Ba,lane 1) followed by 210 copies of gpD-p246-c-myc(31 kDa, Figure 3Ba, lane 2) and 154 copies of gpD-p24-c-myc (39 kDa, Figure 3Ba, lane 3 and Figure3C). In the case of M13, the lane corresponding tophage displaying p241 (lane 1, Figure 3Bb and Bc)showed only one band having molecular mass(,13 kDa as gVIIIp fusion and ,60 kDa as gIIIpfusion) less than calculated for the fusion protein(Figure 3C). Since full-length fusion protein wasnot visible on the blot, the amount of p241 fusionprotein on M13 phage could not be determined.The lane corresponding to M13 phage displaying

Figure 3. Analysis of phage displaying fragments of HIV-1 capsid protein, p24. (A) ELISA: microtitre plates werecoated with ascitic fluid of anti-p24 mAb, H23 (1:1000 dilution) and assay performed as described in Materials andMethods. Phage displaying p241 (amino acid residues 1–72 of p24) as fusion to gVIIIp (A), gIIIp (B) and gpD (F);phage displaying p246 (amino acid residues 1–156 of p24) as fusion to gVIIIp (W), gIIIp (X) and gpD (%); phage dis-playing p24 (amino acid residues 1–231 of p24) as fusion to gVIIIp (K), gIIIp (O) and gpD (i). (B) Western blot:1 £ 108 lambda phage and 1 £ 1011 M13 phage were electrophoresed on 0.1% SDS-12.5% PAG (a and b) or 210% PAG(c), transferred onto PVDF membrane and probed with anti-c-myc mAb, 9E10, followed by HRP-conjugated goatanti-mouse IgG. a, Lambda phage displaying fusion protein with gpD; b, M13 phage displaying fusion protein withgVIIIp; c, M13 phage displaying fusion protein with gIIIp. gIIIp has aberrant mobility (55–60 kDa instead of 45 kDa)accounting for the difference in the calculated and the observed molecular mass. Lane C, phage displaying gpD-c-myc fusion protein; lane 1, phage displaying p241 fused to coat protein; lane 2, phage displaying p246 fused to coatprotein; lane 3, phage displaying p24 fused to coat protein. MW, molecular mass markers with molecular mass inkDa. Arrowhead denotes the degradation product. (C) Quantification of displayed fusion protein on lambda andM13 phage. The number of fusion protein molecules per phage particle was quantified by densitometric scanningof Western blot (developed with anti-c-myc mAb, 9E10, in B using purified fusion protein GST-c-myc as calibrationstandard. MW, calculated molecular mass of fusion protein in kDa. nd, not determined.


p246 and p24 showed two major bands in eachblot (Figure 3Bb and Bc). The band with slowermobility corresponded to the calculated molecularmass of the fusion protein but the second, moreintense band, showed mobility similar to that seenin the lane with M13 phage displaying p241,suggesting these to be degradation products thathad retained the c-myc epitope. This faster movingband was found to be reactive to mAb 9E10 but notto mAb H23, confirming the loss of amino acidsfrom the N terminus (data for mAb H23 notshown). Densitometric scanning showed that M13phage displayed less than two copies of the fusionprotein per phage particle (Figure 3C).

The Western blot data obtained with mAb 9E10correlated well with the ELISA data obtained forreactivity of phage to mAb H23. The full-lengthp241-gVIIIp/gIIIp fusion protein may be presentin extremely low quantities on M13 phage (notdetected in Western blot); however, the degra-dation product that was displayed on the phagesurface retained H23 epitope (confirmed byWestern blot of phages using H23; data notshown) resulting in the high reactivity observed inELISA. This analysis clearly shows that the lambdaphage system is capable of displaying proteins ofdifferent sizes with large domains in much higherdensity than the M13 phage system, with lessdegradation of the fusion protein.

High-density display leads to efficientselection in bio-panning

The utility of high-density display on lambdawas evaluated in epitope mapping of monoclonalantibodies, and polyclonal serum from human sub-jects. For this purpose, a gene-fragment library ofPseudomonas exotoxin A (PE) derived peptides asfusion to gpD of lambda or to the gIIIp of M13was constructed. For making the lambda library,DNA encoding PE-38, a 38 kDa fragment of PE,23

was digested with DNase I to produce 50–200 bprandom fragments that were flushed and ligatedto SmaI (CCCGGG)-digested donor plasmidvector, pVCDcDL3 (Figure 1C). BM25.8 (Creþ)cells were transformed with the ligation sample

and a library efficiency of 1 £ 107 transformantsper microgram of vector DNA was obtained.Analysis of individual transformants showed that98% clones were recombinants and contained PE-derived inserts that were randomly distributedalong the PE sequence (data not shown). This plas-mid library was then infected with lDL1 to allowrecombination and formation of cointegrates. Thelambda library obtained consisted of phages dis-playing 20–70 amino acid residue peptides fusedto the C terminus of gpD and a similar M13 library(made using the protocol as described in Materialsand Methods) comprised of phages carryingPE-derived peptides fused to the N terminus ofgIIIp. Analysis of individual tranductants of thephage library confirmed that the inserts were faith-fully transferred from the plasmid to the lambdagenome during recombination and their distri-bution was random along the PE sequence. Also,the lambda library contained 18% of clones thathad the PE fragment in-frame with the gpD codingsequence and therefore displayed PE-derived pep-tide on the phage surface as against only 5% ofclones of a similar kind in the M13 library. This isbecause C-terminal fusion to gpD in lambda allowsthree times more PE-derived sequences to be in-frame with the gpD sequence and get displayed ascompared to N-terminal fusion to gIIIp in M13.

The lambda and M13 libraries were first used tomap the epitope recognised by a mAb against PE.The binding of lambda phage particles to mAb-coated wells was 2000 times more than the bindingto uncoated wells, while with the M13 library thisratio was only ten times (Table 3). Sequence analysisof individual phage clones obtained in eluatesfrom mAb-coated wells revealed that 88% of theanalysed lambda clones contained the PE fragmentinserted in-frame with the gpD sequence, as com-pared to 63% of M13 clones. This difference reflectsmore specific binding of lambda phages to mAb-coated wells. These individual phage clones werethen tested for binding to anti-PE mAb in ELISA.The lambda clones produced high dose-dependentreactivity in ELISA to anti-PE mAb, while none ofthe M13 clones showed any significant reactivitydespite the addition of 1000-fold more M13 phage

Table 3. Panning of lambda and M13 phage PE gene-fragment library on anti-PE mAb

Lambda library M13 library

Test Control Test Control

Input phage 1 £ 108 1 £ 108 1 £ 1010 1 £ 1010

Output phage 1 £ 104 5 9 £ 105 9 £ 104

Fold enrichmenta 2 £ 103 10% Clones with PE fragment in frame with coat protein

(no. positive/no. analysed)b

88 (22/25) 63 (23/36)

% Clones ELISA reactive (no. positive/no. analysed)c 100 (12/12) 0 (0/14)

Test, coating with anti-PE mAb; Control, no coating.a Fold enrichment ¼ output phage in test/output phage in control.b The inserts in clones from test wells selected after panning were sequenced to check for reading frame of PE fragment with

respect to the coat protein.c The clones displaying PE peptides were analysed for binding to anti-PE mAb in ELISA.


(data not shown). The low density of fusion proteinon M13 phage surface in combination with the lowaffinity of anti-PE mAb might have resulted invery low capture of M13 phages on mAb-coatedwells, which could not be detected in ELISA.These results clearly demonstrate that the high-density display on lambda phage leads to morespecific binding, which results in higher enrich-ment during panning and higher ELISA reactivityof enriched clones.

The libraries were then used to map immuno-dominant epitopes of PE using polyclonal serum.Appearance of anti-toxin antibodies in serum ofpatients undergoing immunotoxin therapy is amajor obstacle in the way of repeated treatmentwith immunotoxin and success of immunotoxinsin cancer therapy. Identification of immuno-dominant epitopes in toxin molecules will enableengineering of an altered toxin, which retains fullbio-efficacy but will not elicit neutralising anti-bodies when administered to patients. For thispurpose, the PE gene-fragment library was usedto identify regions of PE against which antibodiesare present in the serum of human patients aftertreatment with recombinant immunotoxins con-taining PE-38.24 The anti-PE response in thesepatients is characterised by low titre of antibodiesthat have weak affinity, since the patients areadministered few doses of immunotoxin in ashort period of time.

Microtitre wells were coated with pre-treatment(control) and post-treatment (test) sera frompatients administered immunotoxin therapy, andpanning was performed on individual serumsamples. The results obtained were similar for allsamples. As shown in Table 4, for one sample, theenrichment obtained was more for the M13 library(123-fold) as compared to lambda library (20-fold).However, only 16% of the M13 clones obtainedfrom the test sample had the PE fragment in-framewith gIIIp while 90% of the selected lambda cloneshad the PE fragment in-frame with gpD. The DNAsequence of individual phage clones recoveredfrom test wells was translated and aligned to thePE sequence. The alignment data showed that allthe l clones aligned to the last 50 amino acid resi-dues of PE (Figure 4A), clearly indicating thisregion as the epitope. In the case of M13, the clones

aligned to different regions in PE and no commonalignment could be deduced. A few clones alignedto the last 50 residues of PE (Figure 4A) but theirnumber was not sufficient for this region to be

Table 4. Panning of lambda and M13 phage PE gene-fragment library on human serum

Lambda library M13 library

Control Test Control Test

Input phage 1 £ 108 1 £ 108 1 £ 1010 1 £ 1010

Output phage 1 £ 103 2 £ 104 6 £ 104 7 £ 106

Fold enrichmenta 20 123Clones with PE fragment in frame with coat protein %(no. positive/no. analysed)b

25 (8/30) 90 (30/33) 9 (3/34) 15.7 (11/70)

Control and Test refer to wells coated with pre-treatment serum and post-treatment serum of patients, respectively.a Fold enrichment ¼ output phage in test/output phage in control.b The inserts in clones from test wells selected after panning were sequenced to check for reading frame of PE fragment with

respect to the coat protein.

Figure 4. Epitope mapping using anti-PE serum.(A) Alignment of inserts in phage selected after panningof PE gene-fragment library in M13 and lambda phageson serum from PE-immunotoxin-treated patients. Only 15clones (out of 30 clones) of lambda are shown. The continu-ous line represents PE-38 and the numbers above the linedenote amino acid position in the full-length PE. All theclones were tested in ELISA but the clones whose data areshown in (B) are indicated as a to d. (B) Reactivity ofphage displaying PE fragments in ELISA. The details aredescribed in Materials and Methods. Reactivity of M13phage, (a) (A,B) and (b) (W,X) and lambda phage, (c) (K,O)and (d) (L,P) to pre-treatment sera (open symbols) andpost-treatment sera (filled symbols).


demarcated as an immunodominant epitope.A second round of panning with the amplifiedeluate of the first panning of M13 phage did notresult in any increase in the number of clones withalignment in any one region of PE (data notshown). Therefore, the exact epitope(s) againstwhich antibodies are present in serum could notbe deduced from M13 library data. This differencein the results for the two libraries could be due tolow ligand concentration per phage particle in thecase of M13 as well as the presence of a high num-ber of non-displaying clones in the total M13 popu-lation, which resulted in more non-specific bindingand low binding of specific phage in the well.Samples from several patients were used forpanning and similar results obtained (data notshown). ELISA was performed to test the clonesfor reactivity to serum antibodies. In ELISA, thelambda clones showed high dose-dependent reac-tivity with post-treatment serum pool and gavelow reactivity with pre-treatment serum pool(Figure 4B; data for two representative clones areshown). On the other hand, M13 phage clonesshowed reactivity with pre-treatment serum withonly marginal increase in reactivity with post-treatment serum, indicating non-specific bindingof M13 phage to human immunoglobulins coatedin the microtitre well. The difference in ELISAreactivity between lambda and M13 phages wastwo to three orders of magnitude, again establish-ing the importance of high-density display onlambda phage in the identification of immuno-dominant epitopes using polyclonal serum.

The biopanning data described above clearlyshow that high-density display on lambda phageincreases panning efficiency from a library andoffers a distinct advantage over M13 phage inselection of specific binders.

Display of disulphide bond-containing proteins

One major application of phage display tech-nology is identification of protein–protein inter-action cascades wherein a plethora of proteinsequences are displayed on the phage surface,several of which might contain disulphide bondsessential for their function. We used the single-chain fragment (scFv) of an antibody as fusionpartner with gpD to test the display of disulphide-containing proteins in functional form on lambda.An scFv molecule contains two intra-moleculardisulphide bonds, which are essential for itscorrect conformation and activity. Therefore, func-tional display of scFv as gpD fusion on lambdasurface will indicate that disulphide bonds areformed in proteins displayed on lambda.

Mesothelin is a glycoprotein present on the sur-face of cancer cells and is a promising candidatefor targeted therapies.25 SS126 is a high-affinityvariant of anti-mesothelin antibody SS.25 Lambdaphage displaying SS1 scFv (lDcSS1DL1) were pro-duced by recombination as described in Materialsand Methods and purified. These phages display

SS1 scFv fused at the C terminus of gpD with ac-myc tag at the C terminus of scFv. In ELISAon anti-c-myc-coated plates, the binding oflDcSS1DL1 was about 30 times less than that oflDcDL1 (Figure 5A). Thus, lDcSS1DL1 displayedabout 10–15 copies of D-scFv-c-myc fusion proteinin comparison to lDcDL1 that displayed 400copies of D-c-myc fusion protein per phage par-ticle. Functionality of SS1scFv displayed on lambdawas checked by binding of phage to the naturalligand of SS1, mesothelin. lDcSS1DL1 phage wereadded to mesothelin-coated wells and capturedphage detected using anti-lambda phage poly-clonal sera. lDcSS1DL1 phage showed specificdose-dependent binding to mesothelin, indicatingthat the displayed scFv molecules were functional(Figure 5B). lDL1 and lDcDL1 phages that didnot display SS1 scFv showed no binding tomesothelin. Further, 5 £ 109 lDcSS1DL1 phagesgave the same binding to mesothelin as 1 £ 1011

M13 phage displaying SS1 scFv fused to gIIIp

Figure 5. ELISA of lambda phage displaying SS1scFv.Microtitre plates were coated with anti-c-myc mAb 9E10(A), or recombinant mesothelin (B). Purified l DcSS1DL1phage (X), l DcDL1 phage (K) and lDL1 phage (A) wereadded and ELISA performed as described in Materialsand Methods.


(data not shown), indicating that the number offunctional scFv molecules present per lambdaparticle was several-fold more than per M13particle. This was confirmed by Western blot analy-sis using anti-c-myc mAb 9E10. Here, 1 £ 109

lDcSS1DL1 phage showed a band correspondingto gpD-scFv-c-myc fusion protein while a sameintensity band of scFv-c-myc-gIIIp was seen with5 £ 1010 M13 phage displaying SS1scFv fused togIIIp (data not shown). This result establishesthat disulphide bond-containing proteins are alsodisplayed in higher numbers on lambda phage ascompared to M13 phage.

Discussion

Here, we describe a user-friendly lambda-basedphage display system with a simple strategy forhigh-efficiency cloning of foreign DNA intolambda phage genome and display of the encodedpeptide/protein in functional form in high num-bers on the surface of lambda phage fused to theC terminus of the D protein (gpD). In this system,the display of peptides, large protein domainsand full-length proteins, including disulphide-bonded proteins, on the surface of bacteriophagelambda was several orders of magnitude higherthan on the surface of bacteriophage M13(which to date is the most widely used displayvehicle). Also, this high-density display on lambdaphage was extremely useful in mapping epitopesof monoclonal antibodies and in identifyingimmunodominant epitopes of a toxin moleculeusing a toxin gene-fragment library displayed onlambda.

A prerequisite for any surface display system tobe used in the form of a library is efficient cloningof DNA sequences encoding the peptides/proteinsto be displayed, into the genome of the displayvehicle. With a large size of 50 kb, it is not easy toachieve cloning efficiencies in lambda as high asthose obtained with plasmid DNA using currenttransformation protocols. As a result, makinglarge libraries in lambda is an arduous task. Thelambda phage system described here overcomesthis limitation because the cloning of the foreignDNA sequence (encoding the protein/peptide tobe displayed) is carried out in a plasmid vector.Then, by a highly efficient process of phage infec-tion and in vivo recombination, the clonedsequence is integrated into the lambda genomeand the peptide encoded by the cloned sequenceis displayed as gpD fusion protein on the surfaceof progeny lambda phage particles. Because thecloning is done in a plasmid, high transformationefficiencies (by electroporation) can be achieved.Also, the recombination occurs in vivo, eliminatingthe need to isolate lambda DNA, clone DNAsequences into it and package the recombinantlambda in vitro. The frequency of recombinants(with insert) at the plasmid level as well as thephage level (cointegrates) is greater than 90% as

against 3–15% reported for direct cloning inlambda display vectors.27 We have constructed agene-fragment library of the M. tuberculosisgenome in lambda with library size of 3 £ 107

clones per microgram of vector having 90%recombinants (unpublished data).

The recombination in vivo is mediated by loxrecombination sequences present in the donorplasmid and the recipient lambda vector. We haveused two non-compatible lox recombinationsequences, lox Pwt and lox P511;

19 recombination canthus occur only in trans resulting in integration ofplasmid sequence into lambda DNA. This strategycircumvents the problem of excision of integratedplasmid as observed earlier9 due to the presenceof two compatible lox Pwt sites in the cointegrate.The recombinants (cointegrates) formed can beeasily selected for antibiotic resistance conferredby the integrated plasmid. The cointegrates containthe cloned foreign DNA sequence as part of theirgenome and the corresponding phage display theencoded peptide/protein as a gpD fusion proteinon their surface. In the system described here,more than 75% of cointegrates were DCO cointe-grates and are inert to any recombination in cis.The single crossover cointegrates have two lox Pwt

and two lox P511 sites and can undergo recombina-tion in a Cre2 host as observed earlier by otherworkers,9 which can result in either formation of aDCO or loss of integrated plasmid. It should bepossible to eliminate SCO by using a recipientlambda vector having a counter-selectable markerin place of lacZa and selecting for cells containingDCO co-integrates on non-permissive growth med-ium. Another strategy could involve increasing thelength of the sequence (which is excised out duringformation of DCO) between the lox sites in therecipient lambda vector so that, after recombina-tion, in vivo packaging of SCO with genome sizegreater than 51 kb would become inefficient.

The lambda system described here was able todisplay proteins of different sizes (72, 156 and 231amino acid residues), and the number of copies ofeach protein per phage particle on lambda wastwo to three orders of magnitude greater ascompared to display on M13 phages as fusion togVIIIp or gIIIp. The 156 and 231 amino acid resi-due fragments of p24 represent complete func-tional domains of the HIV-1 capsid protein, p24.With such a high density of fusion protein onthe surface, the lambda phage particle can beenvisaged as a dense mass of functional protein,with the encoding DNA encapsulated inside, thatcan be directly used in affinity selection of specificclones from a large library or for studyinglow-affinity protein–protein interactions. Due tolow-density display on M13, the probability of iso-lating low-affinity interacting partners is greatlyreduced. This could be compensated by the avidityeffect due to high-density display on lambda, andthereby the chances of recovering low-affinitypartners in addition to the high-affinity oneswould increase. p24 oligomerises during HIV


capsid formation to form multimers with solutionKd of 1.3 £ 1025 M.28 In an assay to test binding ofp24-displaying phages to recombinant p24,29

lambda phage displaying intact p24 showedapproximately 100-fold better binding to immobi-lised p24 than corresponding M13 phage (unpub-lished results), suggesting that the lambda-basedsystem would be useful in studying low-affinityinteractions. Moreover, gpD not only enables dis-play of few hundred copies of the foreign proteinper phage particle both as N and C-terminalfusion,16,17 but is also expressed at high levels andis present in soluble form in the cell cytosol. gpDhas been shown to have chaperone-like propertiesand is a good fusion partner for expression ofproteins in soluble form.30 This property of gpDmight be responsible for the display of foreign pro-teins in large numbers on the lambda surface andwould also facilitate purification of fusion proteinsequences selected from the lambda displaylibraries.

The high-density display on lambda can beparticularly useful in epitope mapping. Specificepitopes can be identified from complex gene-fragment libraries made from whole genomes ofinfectious viruses and bacteria using immuno-globulins in sera from infected persons as bait.The performance of lambda display in epitopemapping was evaluated using a gene-fragmentlibrary of PE and panning on monoclonal anti-bodies and serum isolated from patients treatedwith PE-based immunotoxins. Here again, thelambda library resulted in specific enrichment andthe selected phages showed higher ELISA reac-tivity than M13, establishing the superiority of thelambda display system. The lambda library wasalso used for epitope mapping of a panel of anti-PEmAbs wherein not only linear epitopes (also deter-mined by a synthetic 20-mer peptide library ofPE) but also larger conformational epitopes wereidentified. These conformational epitopes couldnot be determined by the synthetic peptide libraryof PE (data to be published elsewhere). This high-lights the importance of phage display-basedgene-fragment libraries in epitope mapping. Withpatient’s serum, the M13 system failed to identifyany specific epitope. This can be attributed to lowtitre and low affinity of anti-toxin antibodiespresent in these serum samples, which were notable to bind M13 phages displaying fewer epitopemolecules as compared to corresponding lambdaphage. This high selectivity and sensitivity of thelambda display system would be very useful inrapid identification of immunodominant epitopesin acute viral/bacterial infections where responsein patients may not comprise high-affinity anti-bodies with high titres. Other groups havealso reported more efficient selection of antibodyepitopes using a lambda-based random peptidelibrary in comparison to the M13-based library,8,31

attributing this to the under-representation ofsecretion-incompetent clones in the M13 library.Our data suggest that it may be the large dif-

ferences in display density on lambda and M13that dictate selection efficiencies.

Correct disulphide bond formation can beessential for functional display of large proteindomains in several cases but may not occur in thereducing environment of the cytosol where lambdaassembly takes place. The display of the single-chain Fv fragment (scFv) in functional form onlambda demonstrates that correct disulphide bondformation takes place during cytosolic assembly oflambda particles. It would be difficult to commenton the mechanism of disulphide bond formation;however, cell lysis accompanying lambda phageproduction might be responsible for providingoxidising conditions that enable disulphide bondformation in the molecules displayed on thephage surface. The display of these proteins maybe further improved by using oxidising strains ofE. coli that allow disulphide bond formation in thehost cytosol32 in conjunction with co-expression ofdisulphide bond-promoting chaperones. AlthoughM13 is the system of choice for display of antibodyfragments and their affinity maturation in vitro,functional display of antibodies on lambda inmuch higher numbers than on M13 will be particu-larly useful in isolating low-affinity antibodies,identifying new tumour markers by the abilityto pan with higher sensitivity on cell surfaceswhen the concentration of panning antigen islimited, and in targeted-phage-based therapeuticstrategies.33

Due to the well recognised utility of phage dis-play technology in studying ligand–ligate inter-actions and in identifying a protein/peptideligand by a process of selection rather thanscreening, there has been continual effort toimprove the existing M13-based phage display sys-tem and to develop new systems for higher-densitydisplay of peptides and large protein domains.34 – 36

The new systems are based on large genomephage, mainly T7, T4 and lambda.9,16,17,37 A T7 dis-play system is commercially available for displayof large proteins, however, at low density. The T4display system supports high-density display butthe cloning efficiencies achieved in this system arelow.37 The lambda phage has been shown to dis-play large proteins such as b-galactosidase infunctional form in about 35 copies per phageparticle.16 Here also, the cloning efficiency was notvery high. The system described here has simpli-fied the process of cloning in lambda and the dis-play density achieved for all sizes of proteins isremarkably high.

In summary, the novel approach for cloning anddisplay of peptides/large protein domains in highdensity on phage lambda, described here, shouldbe useful in constructing whole genome librariesfor identifying epitopes using complex polyclonalsera, isolating interacting partners and decipheringprotein interaction networks with high sensitivityand specificity. Cell-targeted delivery and in vivopanning should benefit greatly from this lambdadisplay system.


Materials and Methods

Materials

E. coli strain BM25.8 {supE thiD (lac-proAB) [F0

traD36proAþBþlacIqZDM15 ]limm434 (kanr) P1 (Cmr) hsdR(r2mþ) (Novagen, Madison, WI) was used as Creþ hostfor in vivo recombination. E. coli strain TG1 (supED(hsdM-mcrB)5(rk

2mk2McrB2)thiD(lac-proAB) [F0traD36,

LacIqD(lacZ)M15 ]) was used as Cre2 host for titreingphage lysates and amplification of phages. lDam imm21nin517 was used for constructing lDL1. Collagenase wasobtained from Roche Diagnostics, Germany. Anti-c-mycmAb, 9E10 was produced using hybridoma obtainedfrom ATCC, Manassas, VA. Anti-p24 mAb, H23 wasproduced in-house and its epitope mapped (amino acidresidues 56–66 of HIV-1 p24) using a phage display-based gene-fragment library.7 GST-c-myc was producedin E. coli and purified to homogeneity by affinitychromatography.7 mAbs to PE were raised by immuni-sing mice with a derivative of PE-38 carrying mutationin the active site. The human sera were obtained frompatients undergoing immunotoxin therapy and collectedafter informed consent. For the present study, thesesamples were used as anonymous samples. HRP-conju-gated antibodies were obtained from Jackson Immuno-Research Laboratories, West Grove, PA.

Construction of donor plasmid vectors

The donor plasmid vector, pVCDcDL1, was assembledby ligating the following three segments of DNA bearingcompatible ends. One segment was prepared by PCR-based amplification of the lambda D gene to create aHindIII site before the Shine–Dalgarno sequence and toincorporate after the last codon of D gene, a sequenceencoding spacer (GGSG), followed by a collagenasecleavage site (PVGP), NheI site, ten codons of a stuffersequence, codons for decapeptide tag, c-myc, stopcodon, and SalI and EcoRI restriction sites. Theassembled PCR product was digested with HindIII andEcoRI to obtain a 475 bp fragment. The second segmentwas also assembled by PCR and contained the origin ofreplication of filamentous phage ( fori) flanked by thesequence for restriction site SstI and lox P511

19 on oneend and the sequence for lox Pwt and an EcoRI restrictionsite on the other end. The product was digested with SstIand EcoRI to obtain a 515 bp fragment. The third seg-ment formed the backbone of the plasmid vector. Forthis, an SstI restriction site was created by site-directedmutagenesis in pUC119 upstream of the b-lactamasegene to produce a plasmid pUCSSt. pUCSSt wasdigested with HindIII and Sst I and dephosphorylated toobtain a 2.5 kb fragment. pVCDcDL1 (GenBank acces-sion no. AY10049), was obtained from ligation of thethree fragments, and sequenced between HindIII andSstI sites using the dideoxy chain termination method.

pVCDcDL3 (GenBank accession no. AY190304) wasconstructed by cloning a cassette encoding the lac promo-ter, RBS and the first 145 codons of lacZ flanked by SmaI/SrfI sites, as NheI–EcoRI insert in pVCDcDL1 (Figure 1C).

Construction of recipient lambda vector

A DNA segment comprising the lac promoter, RBSand first 58 codons of lacZ flanked by sequence for loxP511 and lox Pwt was assembled by PCR to have XbaIcompatible ends and ligated in the unique XbaI site in

lDam at map co-ordinate 24508. The ligation mix wasthen packaged in vitro using the Gigapack II system(Stratagene, La Jolla, CA). The phage mixture producedafter packaging was plated on lawn cells (E. coli strainTG1). The plaques obtained were analysed for recombi-nants by PCR using primers L1 and L4, which flank theXbaI site in lambda (Figure 1B). The recombinantobtained was named lDL1.

Generation of lambda cointegrates andphage production

BM 25.8 cells (Creþ) and TG1 cells (Cre2) transformedwith donor plasmid (carrying foreign DNA) weregrown to A600 nm , 0:3 in LBAmp (LB medium contain-ing ampicillin at 100 mg/ml) at 37 8C. Cells (1 £ 108)were harvested and suspended in 100 ml of lDL1 phagelysate at an MOI of 1.0. After incubation at 37 8C for tenminutes, the sample was diluted in 1 ml of LBAmp con-taining MgCl2 (10 mM) and grown at 37 8C with shakingfor three hours for lysis. For large-scale recombination,the number of cells and the volume of lDL1 wereincreased proportionately to maintain an MOI of 1.0.The cell-free supernatant was used to infect an exponen-tial phase culture of TG1 and Ampr colonies obtained.These Ampr colonies are immune to superinfection andcarry the phage as plasmid cointegrates. The Ampr col-ony containing the lambda cointegrate was grown inLBAmp at 37 8C for four hours. Lambda phage are spon-taneously induced in these cultures and result in com-plete lysis. This cell-free supernatant was then used toinfect TG1 cells to obtain plaques. Phage obtained fromsingle plaques were amplified by the liquid lysis methodat an MOI of 0.01 to obtain lysate with a titre of 5 £ 109

pfu per ml. These phage were further amplified by theliquid lysis method and purified by PEG–NaCl precipi-tation and differential sedimentation.

Construction of lambda and M13 vectors for displayof various fragments of p24

DNA sequences encoding different fragments of HIVcapsid protein p24 were amplified from pVCp2421029

and cloned between NheI–MluI sites to replace thestuffer fragment in pVCDcDL1 and create donor plas-mids pVCDc(p241)DL1/pVCDc(p246)DL1 and pVCDc(p24)DL1. E. coli strain BM25.8 was transformed witheach plasmid and recombination carried out by infectingcultures of each transformant with lDL1 phage to obtainDCO cointegrates of lDc(p241)DL1, lDc(p246)DL1 andlDc(p24)DL1 as described above.

DNA encoding different p24 fragments were alsocloned as Nhe I–Mlu I inserts into phagemid gIII displayvector, pVC3TA726,38 and a similar phagemid gVIIIdisplay vector, pVCp240518426 (V.K.C., unpublishedresults) to obtain various phagemid constructs to pro-duce phage displaying protein fused to gIIIp and gVIIIpof M13, respectively. The M13 phage displaying proteinswere produced by using VCS M13 as described.39

The lambda and M13 phage were purified from cell-free supernatant by PEG precipitation followed byultracentrifugation.

Construction of Pseudomonas exotoxin (PE) gene-fragment library in l and M13 vectors

Random fragments (50–200 bp) of DNA encodingPE-38, a 38 kDa fragment of PE,23 were produced by


DNase I digestion and ligated as blunt-ended fragments(1 mg) in SmaI (CCCGGG)-digested pVCDcDL3 (500 ng)in the presence of restriction enzyme Srf I (GCCCGGGC)using previously described protocols.40 The ligation mixwas electroporated into BM25.8 cells and plated on150 mm LBAmpGlu (LBAmp medium containing 1%glucose) plates to obtain 5 £ 106 independent clones.The transformants were scraped and cell suspensionstored at 270 8C. An aliquot of stored cell suspension(1 £ 108 cells) of the library was grown in 10 ml ofLBAmpGlu to an A600 of 0.3. The cells were harvestedand suspended in 1 ml of lDL1 phage lysate at an MOIof 1.0. After incubation at 37 8C for ten minutes, thesamples were diluted in 10 ml of LBAmp containingMgCl2 (10 mM) and grown at 37 8C with shaking forthree to four hours until cell lysis. The cell-free super-natant (10 ml) was used to infect an exponential phaseculture of TG1 cells (10 ml) at 37 8C for ten minutes andthe cell suspension was plated on 20 LBAmpGlu150 mm plates. The Ampr colonies harbouring cointe-grates were scraped and stored at 270 8C. Cells (1 £ 109)harbouring cointegrates were diluted into 50 ml ofLBAmp medium and grown at 37 8C for eight hours toproduce phage particles. The cell-free supernatant con-taining phage particles was directly used for affinityselection.

PE-derived 50–200 bp DNA fragments were alsoligated to SmaI-digested phagemid-based gIIIp displayvector, pVCEPI13426 (V.K.C., unpublished results) toobtain the gene-fragment library in M13. A library of6 £ 106 independent clones was obtained in TG1 cellsand used to produce M13 phage displaying peptides asdescribed.39

Construction of scFv displaying lambda phage

DNA encoding the scFv fragment of the anti-mesothelinantibody, SS1 was PCR amplified using pPSC7-1-126 astemplate and cloned as an NheI–MluI insert inpVCDcDL1, to obtain donor plasmid pVCDcSS1DL1.BM25.8 cells were transformed with pVCDcSS1DL1 andrecombination performed using lDL1 as describedabove to isolate a clone harbouring DCO cointegrate,lDcSS1DL1. A single colony harbouring DCO cointe-grate was grown in LBAmp at 37 8C for four to sixhours for lysis to occur. The supernatant was used togrow more phage by the liquid lysis method in LBmedium by infecting TG1 cells at MOI 0.01. Phagefrom cell-free supernatant were purified by PEG–NaClprecipitation and differential sedimentation.

Estimation of phage binding and affinity selection ofbinders by bio-panning

To check the presence of binder phage, wells of micro-titre plates (Maxisorp, Nunc, Rochester, NY) were coatedwith 1:1000 dilution of ascitic fluid of anti-c-myc mAb9E10 and phage lysate was added to the coated wells40

and incubated for one hour at 37 8C. The unboundphage were removed by washing. To assay the capturedlambda phage, 0.3 ml of exponential phase TG1 cellswere added to each well and incubated for ten minutesat 37 8C. Cells were then removed and serial dilutionsplated to determine phage-infected cells as pfu and cfu.The pfu and cfu indicate the number of phage bound tothe coated wells.

For panning of the PE gene-fragment library on mAb,wells were first coated with goat anti-mouse IgG (Fc frag-

ment-specific) antibody followed by 1:100 dilution ofanti-PE mAb culture supernatant (Test wells) or buffer(Control wells). For panning of the PE gene-fragmentlibrary on human serum, wells were coated with goatanti-human (IgG þ IgM, Fc fragment-specific) antibodyfollowed by 1:100 dilution of serum from patients treatedwith PE-based immunotoxins (Test wells) or pre-treat-ment serum of patients (Control wells). Phage lysate(1 £ 108 phages per well for lambda library and 1 £ 1010

phage per well for M13 library) was added to each well,incubated at 37 8C for one hour and unbound phagesremoved by washing. For the M13 library, the capturedphage were eluted using low-pH buffer40 and titratedon TG1 as cfu. In the case of lambda phage, one unit ofcollagenase in 0.1 ml of phosphate buffer (20 mM, pH7.4) was added to each well for ten minutes at room tem-perature. The released phages were titrated on TGI toobtain Ampr colonies. Individual Ampr colonies weregrown and phage particles produced as described pre-viously by infecting with helper phage for M13 clones39

and by growing colonies in LBAmp medium till com-plete cell lysis for lambda phage clones. The cell-freesupernatants were subsequently used for ELISA.

Western blot analysis and ELISA of phage

For Western blots, purified phage were electrophor-esed under reducing conditions on 0.1% (w/v) SDS/10% or 12.5% (w/v) PAG followed by electroblottingonto PVDF membrane (Immobilon, Millipore, Bedford,MA). Fusion proteins were detected with 1:1000 dilutionof ascitic fluid of anti-c-myc mAb, 9E10/anti-p24 mAb,H23 followed by horse radish peroxidase (HRP)-conju-gated goat anti-mouse IgG (H þ L) antibody.

For ELISA, wells of Maxisorp plates (Nunc, Rochester,NY) were coated with 1:1000 dilution of ascitic fluid ofmAb 9E10/H23 and purified phage were added to thecoated wells. The bound phages were detected with rab-bit anti-lambda polyclonal serum or rabbit anti-M13polyclonal serum followed by HRP-conjugated goatanti-rabbit IgG (H þ L) antibody.

Binding of phages produced by individual clonesselected in bio-panning was tested in ELISA. For this,wells were coated with 1:1000 dilution of rabbit anti-lambda polyclonal serum or rabbit anti-M13 polyclonalserum and corresponding phages were added to thecoated wells. After removing unbound phage, 1:100dilution of anti-PE mAb (culture supernatant) or serumfrom patients treated with PE-based immunotoxins wasadded. The bound phage were detected with HRP-conjugated goat anti-mouse IgG (H þ L) antibody orHRP-conjugated goat anti-human (IgG þ IgM) antibody.

For ELISA of phages displaying SS1 scFv, microtitrewells were coated with 100 ng of recombinantmesothelin.26 After blocking the unoccupied sites with2% non-fat dry milk, purified lambda phage wereadded to the coated wells and incubated at 37 8C forone hour. The unbound phage were removed by wash-ing and the bound phage detected with rabbit anti-lambda polyclonal serum followed by HRP-conjugatedgoat anti-rabbit IgG (H þ L) antibody.

Acknowledgements

The authors are grateful to Dr Ron Hoess for


providing l Dam vector for this work. We thankAbhishek Kulshreshta for help in panning of theM13 phage-based PE library. Dr J. P. Khurana isacknowledged for critical review of the manu-script. The Department of Biotechnology, Govern-ment of India, financially supported the work.

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Edited by M. Gottesman

(Received 4 June 2003; received in revised form 29 August 2003; accepted 15 September 2003)


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