Journal of Immunological Methods 321 (2007) 164–173www.elsevier.com/locate/jim

Research paper

Isolation of trans-acting genes that enhance soluble expression ofscFv antibodies in the E. coli cytoplasm by lambda phage display

Raphael Levy a, Ian J. Molineux a,b, Brent L. Iverson a,c, George Georgiou a,b,⁎

a Institute for Cell and Molecular Biology, University of Texas, Austin, United Statesb Section of Molecular Genetics and Microbiology, University of Texas, Austin, United States

c Chemistry and Biochemistry, University of Texas, Austin, United States

Received 26 July 2006; received in revised form 10 January 2007; accepted 18 January 2007Available online 20 February 2007

Abstract

Functional antibody fragments with native disulfide bonds can be expressed in Escherichia coli trxB gor mutant strains havingan oxidizing cytoplasm that allows the formation of disulfide bonds. However, expression yields in the cytoplasm are generallylower than those obtained by secretion into the periplasm. We developed a novel methodology for the screening of genomic DNAfragments that enhance expression yields of scFvs in the cytoplasm of trxB gor cells by capitalizing on bacteriophage lambdadisplay. The anti-digoxin 26.10 scFv was displayed on λ as a fusion to the coat protein gpD. A genomic E. coli library was clonedinto λgt11 downstream from the lac promoter and used to lysogenize cells transformed with a plasmid encoding the scFv–gpDfusion. Following induction of expression of the cloned gene fragments, phage was prepared and screened for improved functionaldisplay via panning against immobilized hapten. Phage exhibiting improved display was isolated after two rounds. One of theisolated clones, encoding the N-terminal domain of the alpha-subunit of RNA polymerase (α-NTD), was shown to increase theyield of scFv expressed in soluble form in the cytoplasm.© 2007 Elsevier B.V. All rights reserved.

Keywords: Bacteria; Cytoplasm; Folding modulators; Lambda

Abbreviations: ELISA, enzyme-linked immunosorbent assay;IPTG, isopropyl-β-D-thiogalactopyranoside; OPD, o-phenylenedia-mine; scFv, single-chain Fv; PCR, polymerase chain reaction; LB,Luria–Bertani; BSA, bovine serum albumin; OD, optical density;PBS, phosphate buffer saline; M.O.I, multiplicity of infection; HRP,horseraddish peroxidase; PEG, polyethylene glycol; X-GAL, 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside; FAB, antigenbinding fragment.⁎ Corresponding author. Institute for Cell and Molecular Biology,

University of Texas at Austin, Austin, TX 78712–1064, United States.Fax: +1 512 471 7963.

E-mail address: ggmjd@mail.utexas.edu (G. Georgiou).

0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2007.01.017

1. Introduction

Expression of antibody fragments in E. coli is typ-ically achieved by secretion into the periplasmic space,an oxidizing subcellular compartment which allows theformation of disulfide bonds that are required for thestability of the molecule (Segatori et al., 2006; Wornand Pluckthun, 2001). The yield of periplasmicallyexpressed antibody fragments is highly dependent on theprotein sequence. While some secreted FAB and scFvfragments can be produced at the gram scale in fer-menters, other antibodies are refractile to expressionbecause of toxicity or protein aggregation problems

165R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

(Carter et al., 1992; Hayhurst and Harris, 1999; Hayhurstet al., 2003; Horn et al., 1996).

The toxicity of secreted proteins is typically associ-ated with the jamming of the secretory apparatus and cantherefore be prevented if the protein is expressed in thecytoplasm. In addition, in contrast to the bacterial peri-plasm, the cytoplasm contains an extensive chaperonenetwork that exploits ATP hydrolysis for substrate bind-ing or release and that can be exploited for the preventionof recombinant protein aggregation. However, the cyto-plasm is normally maintained in a strongly reducing statethat prevents the formation of disulfide bonds throughthe action of the thioredoxins–thioredoxin reductase(encoded by trxA,trxC,trxB), glutathione–glutathionereductase (gor) and glutaredoxin (grxA-C) pathways(Ritz and Beckwith, 2002). Mutational inactivation ofone or more of these pathways renders the cytoplasmprogressively more oxidizing (Derman et al., 1993; Ritzand Beckwith, 2002). The highest level of cytoplasmicprotein oxidation is observed upon inactivation of boththe thioredoxin and glutathione reduction pathways intrxB gor mutant strains (Bessette et al., 1999). Bothsingle-chain Fvs (which contain 2 disulfides) and FABfragments (5 disulfides) have been successfully expressedin functional formwithin the cytoplasm of trxB gor E. colicells (Heo et al., 2005; Levy et al., 2001; Monje-Casaset al., 2001). Unfortunately, the yield of active proteinobtained in these studies was not higher than thosetypically obtained in the periplasm (Philibert andMartineau, 2004). Expression was shown to be enhancedby gene fusions to solubilizing partners such asthioredoxin, or by co-expression of proteins involved infolding, namely chaperones and disulfide isomerases(Jurado et al., 2006; Levy et al., 2001). In the latterinstance there is no way to predict a priori whether aparticular chaperone or some other gene product will aidsoluble expression. Furthermore, extensive experimenta-tion may be required to determine the precise reasons forlow soluble protein yield and to determine whatchaperones or other factors might need to be co-ex-pressed. Recently, Philibert and Martineau (2004)attempted to exploit α-complementation of β-galactosi-dase for the directed evolution of scFvs exhibitingimproved cytoplasmic expression. Unfortunately, thisscreen did not yield better expressed mutants.

Filamentous phage has been exploited for the screen-ing of libraries to isolate genes that enhance the display orimprove the folding of a polypeptide displayed as a fusionto the infectivity protein p3 (Bothmann and Pluckthun,1998; Lafond et al., 2003). In this approach, a libraryexpressed from an inducible promoter is cloned onto aphagemid. The p3 fusion of the displayed polypeptide is

expressed from the same phagemid or from a secondplasmid. Following infection with helper phage, the p3fusion is incorporated into the virion which packages thephagemid DNA. Within a particular cell, phagemid-encoded proteins that enhance expression result inimproved display of the p3 fusion. In this manner, thedegree of display becomes physically linked to thephagemid gene whose product affects the expression and/or folding of the p3 fusion protein. This strategy was firstused by Pluckthun and coworkers for the isolation of theperiplasmic chaperone Skp which enhances the expres-sion of several scFvs (Bothmann and Pluckthun, 1998;Hayhurst and Harris, 1999). In later studies phage displaywas used to enrich mutants of disulfide isomerase DsbCthat act catalytically in the E. coli periplasm to assist thefolding of the multi-disulfide protein tissue plasminogenactivator fused to p3 (Lafond et al., 2003).

Filamentous phage assembles in the periplasm andtherefore cannot be employed for the isolation of genesthat affect the expression of scFvs in the cytoplasm. Incontrast to M13, the biogenesis of the T7 and λ capsidsoccurs in the cytoplasm and these viruses have beenexploited for library screening applications (Danner andBelasco, 2001; Sternberg and Hoess, 1995). Successfuldisplay of heterologous proteins on phage λ has beenaccomplished using the tail protein gpVor the minor coatprotein gpD (Dunn, 1995; Maruyama et al., 1994).However, a major disadvantage of gpV is that only a fewcopies of displayed protein are tolerated on the phage λtail (Hoess, 2002). By contrast, peptides and even largeproteins can be displayed polyvalently at the N- or C-terminus of gpD. Polyvalent display is advantageous inthe isolation of polypeptides that exhibit low ligandbinding affinity (Gupta et al., 2003; Mikawa et al., 1996;Santini et al., 1998; Sternberg and Hoess, 1995). SeveralcDNA libraries displayed on λ phage via gpD have beenaffinity-selected on antibodies or other ligands (Ansuiniet al., 2002; Beghetto et al., 2001; Santi et al., 2000;Santini et al., 1998; Zucconi et al., 2001). Similarly, λdisplay has been employed for epitope mapping and bothprotein:protein and protein:DNA interaction analyses(Cicchini et al., 2002; Hagiwara et al., 2002; Moriki et al.,1999; Stolz et al., 1998).

We report here the development of a system for theisolation of phage encoded genes that act in trans to assistthe expression of a cytoplasmically expressed scFv intrxB gor E. coli. The anti-digoxin 26.10 scFv antibody(Francisco et al., 1993) was displayed as a fusion to the N-terminus of the viral coat protein gpD. We show that λdisplaying functional scFv antibody can be produced inE. coli trxB gor cells and that this phage can be enrichedfrom λ displaying an unrelated antibody. A library of E.

166 R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

coli gene fragments was inserted downstream from thelac promoter in λ gt11, which was then lysogenizedinto E. coli trxB gor cells. Following two rounds ofenrichment on hapten we isolated a gene fragment thatconferred improved display and increased the amount ofactive 26.10 scFv expressed alone, without fusion to thegpD protein.

2. Materials and methods

2.1. Strains

E. coli DR473H was constructed by P1 transductionof strain DR473 (MC1000 phoA(PvuII) phoR malF3ΔtrxB gor552Tn10 ahpC⁎) (gift from J. Beckwith) withthe ΔhflKC::Km from E. coli AK990 (kindly providedby Dr. Kihara (Kihara et al., 1996). The hfl mutationincreases the stability of the λ cII protein, resulting in aDR473H lysogenization efficiency of about 90%. Y1088[F−Δ(lac)U169 glnV supF hsdR (rk

−mk+)metB trpR

fhuA21 proC::Tn5 (pMC9; TetrAmpr)] (Huynh, 1985)was provided by Stratagene (La Jolla, CA) andBL21 (ompT gal dcm, hsdSB(rB

−,mB−)) (Novagen, WI)

was described by Studier et al. (1990).

2.2. Construction of scFv–gpD fusions

The gene encoding anti-digoxin scFv (26.10)was amplified by PCR from the expression plasmidpET25b-scFv (Burks et al., 1997; Chen et al., 1999;Panka et al., 1988) with the external forward primer T7Promoter (5′-TAATACGACTCACTATAGGG-3′) (Nova-gen, WI) and the reverse primer 3′scFvHsVL (5′-CATGAACATACCGGAGCCACCGCGGGA-ATCCTCGGGGTCTT CCGGG-3′). The reverse primerintroduced the gene fragment encoding the linker between26.10 and gpD. The linker sequence encoding the aminoacid sequence SRGGSGMFM was previously used (Yanget al., 2000). Similarly, the gene encoding the minor coatprotein gpD of phage λ was amplified by PCR fromλ cI857Sam7 , us ing primers 5 ′FABLD (5 ′-TCCCGCGGTGGCTCCGGTATGTTCATGACGAG-CAA AGAAACCTTT-3′) and 3′gpDstop (5′-GAAT-TAAGCTTAGATGAATTCACTATTA AACGATGCT-GATTGC-3′). Subsequently, the 26.10 and the gpD PCRproducts were used as templates for an overlap extensionPCR, employing the external primers T7 Promoter and 3′gpDstop, in order to generate the gene fusion 26.10 scFv–gpD. The gene encoding the fusion protein was purified,digested with NdeI and HindIII and ligated to theexpression plasmid vector pAR3-skp (Levy et al., 2001)to yield pAR-26.10 scFv–gpD. Similarly, the gene

fragment encoding the fusion protein between gpD and1Hhum scFv, a humanized antibody that binds to the PAcomponent of the B. anthracis toxin, was amplified byPCR from the plasmid pMopac16–1HhumscAb (Hayhurstet al., 2003) with the forward primer 5′HumscFv (5′-GTATGTTAGATAGATTTAATTG CGGCCGCCCGAG-GAGACGGTGACCAGGGTTCC-3′) and the reverseprimer 3′HumscFv (5′-GTATGTTAGATAGATTTAA-TTGCGGCGCCCGAGGAGACGGT GACCAGGG-TTCC-3′). The DNA was then digested with NdeIand NotI, and ligated in the place of the 26.10 scFvgene fragment in the plasmid pAR-26.10 scFv–gpD inorder to generate the expression plasmid pAR-1HhumscFv–gpD.

2.3. Construction of an E. coli genomic DNA library in λ

Genomic DNA from the E. coli strain BL21 wasisolated with the DNeasy Tissue Kit (Qiagen, CA). 8 μgDNA were partially digested with Tsp509I, 1000–3000 bp fragments were gel purified and ligated intoEcoRI-digested and dephosphorylated λgt11 (Strata-gene, CA). The ligation mixture was added to packagingextract (Gigapack, Stratagene, CA), briefly centrifuged,and incubated at room temperature for 1.5 h. The as-sembly reaction was terminated with the addition of500 μl SM buffer and 0.2% v/v chloroform. Supernatantwas titered, amplified and used for lysogenization ofE. coli DR473 (pAR-26.10 scFv–gpD) following theprotocol of Slominska et al. (1999) and the Lambda InchManual (Boyd et al., 2000).

2.4. Enrichment for lambda phage displaying a specificscFv

The bla gene encoding β-lactamase was amplifiedby PCR together with its upstream constitutive pro-moter from the expression vector pBAD24 (Guzmanet al., 1995), using 5′BLAECORI (5′-TTCTAACT-TATGAATTCTAATTAATTAAGGCTGT TTTGG-CGGATGAGAGAGAAGATT-3′) and 3′BLAECORI(5′-TAAGATATAATAG AATTCTGATTATTAC-CAATGCTTAATCAGTGAGGCACCTATC-3′). ThePCR product was digested with EcoRI and clonedinto λgt11 to give λgt11-bla; a lysogen was then madein DR473H (pAR-26.10 scFv–gpD). Similarly, pRheo(Stratagene, CA), which does not encode a protein, wascloned into λgt11, giving λgt11-pRheo and a lysogenmade in DR473H (pAR-1Hhum scFv–gpD).

Overnight cultures were subcultured to an OD600∼0.5and the expression of scFv–gpD fusion protein wasinduced with 0.2% arabinose for 3 h at 30 °C. Cell pellets

Fig. 1. Amino acid and DNA sequences of the 26.10 scFv–gpD fusion protein. The 26.10 scFv is shown in lower case and the λ phage gpD coatprotein in capital letters. The linkers joining the 26.10 VH with VL and 26.10 with gpD are highlighted in light and dark grey color, respectively. TheHSV-Tag is underlined and stop codons are depicted by asterisks.

167R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

were resuspended in LB medium with 10 mM MgSO4,and transferred first to 42 °C and later to 37 °C in order topermit phage growth. Phage displaying 26.10 scFv or1Hhum scFvwas precipitated with PEG-6000 and titered.A 40:1 ratio of λ phage displaying the 26.10 scFv(4×109 pfu/ml) to phage displaying the 1Hhum scFv(108 pfu/ml) was generated and added to an immunotube(MaxiSorp, Nalge Nunc International, NY) coated with2 μg ml−1 PA antigen (List Biological Laboratories, CA),or to a second immunotube which was coated with

4 μg ml−1 BSA–digoxin. Both immunotubes had beenblocked with 5% milk in PBS overnight at 4 °C.Following rotation at room temperature for 2 h, theimmunotubes were washed 20 times with PBS containing0.05% Tween-20 and 10mMMgSO4. E. coliY1088 cellswere added and infected cells plated. Single plaques wereexcised, submerged in 500 μl SM buffer for 2 h at roomtemperature, and then 2 μl of the phage suspension wereused for PCR amplification of the insert in order to detectthe presence of bla or pRheo.

Fig. 2. Expression of soluble 26.10 scFv–gpD fusion and 26.10 scFvin E. coli DR473 cells. The amount of 26.10 scFv–gpD fusion proteinin cytoplasmic extracts was determined by ELISA, using anti-mouse-IgG [(F(ab′)2 specific] or λ phage-specific polyclonal anti-sera. Datarepresent the average of two measurements with cell lysates from twoindependent cultures.

168 R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

2.5. Library screening

The library of DR473H (λgt11, pAR-26.10 scFv–gpD) lysogens was grown at 30 °C in LB with 10 mMMgSO4 and 34 μg ml− 1 chloramphenicol to anOD600∼0.5. Expression of gene fragments under thecontrol of the λgt11 lac promoter was induced with1 mM IPTG for 30 min prior to addition of 0.2%arabinose for induction of the 26.10 scFv–gpD fusion.After 2.5 h at 30 °C, cells were rapidly harvested bycentrifugation and resuspended in 25 ml LB plus10 mM MgSO4. The culture was then transferred to42 °C with subsequent incubation at 37 °C for 3 h. Afterlysis with chloroform, phage was concentrated andtitered, and panning was carried on immunotubes coat-ed with BSA–digoxin as described above.

2.6. Immunological methods

ELISAassays onmicrotiterwells coatedwith 4μgml−1

BSA–digoxin were performed essentially as described(Levy et al., 2001).

For phage ELISAs, cultures of DR473(λ) (pAR-26.10 scFv–gpD), DR473(λ) (pAR-26.10) and DR473(λ), were grown in LB/10 mM MgSO4 at 30 °C to anOD600∼0.5, and induced with 0.2% arabinose for 2.5 hat 30 °C and then transferred to 42 °C and then to 37 °Cfor 3 h to allow λ lytic growth. Undiluted and 1:2 dilutedsamples were added to ELISA wells, and bound 26.10scFv displaying phages were detected with either rabbit-anti-mouse-IgG [(F(ab′)2 specific] primary anti-sera(Pierce, IL), or rabbit polyclonal anti-λ primary anti-sera (generously provided by Dr. Adhya, NCI, MD).

To estimate the amount of 26.10-displaying λcI857Sam7 phage bound to digoxin, ELISA wells were firstcoated with BSA–digoxin and then blocked with 5%milk/PBS, as previously described. After 1 h incubation atroom temperature and washing, E. coliY1088 was addedand infection was allowed for 30 min at 37 °C. Titers ofphage bound to BSA–digoxin were compared with thosefrom phage that were incubated with 4 μg ml− 1 unrelatedhapten conjugates (BSA–TNBS and ovalbumin–TNBS)(Hermanson, 1996), unrelated carrier proteins (ovalbuminand ovalbumin–digoxin that was prepared as BSA–digoxin) (Smith et al., 1970), or BSA alone.

3. Results

3.1. Display of scFv on λ

The 26.10 scFv binds to digoxin and related cardiacglucosides with nanomolar affinity (Chen et al., 1999;

Francisco et al., 1993). A fusion comprising of the 26.10scFv gene followed by a sequence encoding a shortpolypeptide linker (SRGGSGMFM) (Yang et al., 2000)and then the gpD gene was constructed by overlapextension PCR and placed downstream of the arabinosepromoter in vector pAR3 (Perez-Perez and Gutierrez,1995). The amino acid sequence of the fusion protein isshown in Fig. 1. The resulting plasmid pAR-26.10 scFv–gpD was transformed into E. coli strain DR473 carryingmutations in the trxB and gor genes that render thecytoplasm oxidizing as well as a suppressor mutation,ahpC⁎, required for normal growth in minimal and richmedia. Following induction with arabinose, the cellsproduced functional 26.10 scFv–gpD fusion protein thatbound to digoxin–BSA on ELISA plates and reactedwith both anti-FAB and anti-λ antibodies. As expected,26.10 scFv alone was not recognized by anti-λ anti-bodies (Fig. 2). The 26.10 scFv–gpD fusion gave ahigher ELISA signal than unfused scFv, an effect mostlikely related to the ability of gpD to increase the solu-bility of fusion proteins (Forrer and Jaussi, 1998).

Phage λcI857Sam7 (Wu and Taylor, 1971) carries athermolabile repressor, which is inactive at 42 °C, and adefective holin, which allows the accumulation of phageparticles that are unable to lyse the host cell without theaddition of an external agent (e.g. chloroform). E. coliDR473 was lysogenized with λcI857Sam7 and trans-formed either with plasmid pAR-26.10 scFv–gpD orpAR-26.10. Expression of 26.10 scFv–gpD or the scFv26.10 was then induced by adding arabinose andsubsequently lytic growth was triggered by briefly

Table 1Biopanning of lambda phage displaying 26.10–gpDa

Coated proteins (or proteinconjugates) (400 ng/ELISAplate well)

Eluted phage (pfu/ml)(titer averages fromtwo dilutions)

BSA–digoxin 3.8×108

BSA–TNBS 1.7×107

BSA 2.0×107

Ovalbumin–digoxin 2.7×108

Ovalbumin–TNBS 5.4×106

Ovalbumin 1.3×107

a The titers of lambda phage cI857Sam7, formed in E. coli DR473lysogens expressing 26.10–gpD (from plasmid pAR-26.10–gpD),were determined via biopanning from ELISA plate wells coated withdifferent proteins or protein conjugates.

169R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

transferring the cells to 42 °C. Phage was precipitated,titered and 2.4 × 1010 pfu/ml were incubated onmicrotiterwells coated with BSA–digoxin. Phage prepared fromDR473 (λcI857Sam7) that had been transformed withpAR-26.10 scFv–gpD bound to BSA–digoxin and couldbe detected specifically with anti-λ antibodies resulting ina strong ELISA signal. In contrast, phage prepared fromcells expressing the soluble 26.10 scFv gave a back-ground ELISA signal (Fig. 3).

The amount of λcI857Sam7 phage displaying 26.10scFv that was bound to BSA–digoxin could be estimatedby titering. Titers were about 20-fold higher than whenmicrotiter wells were coated with an unrelated haptenconjugate or with carrier protein alone (Table 1).

3.2. Enrichment

Phage displaying a particular scFv could be enrichedfrom phage displaying an unrelated antibody fragmentby panning on immunotubes coated with antigen.1Hhum scFv is a humanized antibody fragment derivedfrom M18 (Harvey et al., 2004) that binds to the Pro-tective Antigen of the B. anthracis toxin with highaffinity. To distinguish between phage displaying dif-ferent scFvs, specific tags were inserted into thegenome of λgt11 (Young and Davis, 1983). pRheo, a2.8 kb DNA fragment that does not encode any protein,was cloned into λgt11 that was then used to display the1Hhum scFv. The 26.10 displaying phage was taggedby inserting a 1.6 kb DNA fragment encoding the blagene.

Phage isolated from cells expressing the 1HhumscFv–gpD or the 26.10 scFv–gpD fusions was titered

Fig. 3. Phage ELISA analysis of λ displaying 26.10 scFv–gpD. Phageprepared from cells expressing the 26.10 scFv–gpD fusion was boundto digoxin–BSA-coated plates and detected by rabbit polyclonal anti-λanti-sera (lane 1). Phage lysates fromDR473 (λ) cells expressing 26.10scFv alone (lane 2) or DR473 (λ) that do not express any heterologousprotein (lane 3) were used as controls. Dilutions were performed in 1%milk/PBS buffer.

and the display of the respective scFvs was confirmedby ELISA (data not shown). Phage displaying the1Hhum scFv was mixed with a 40-fold excess ofphage displaying 26.10. The phage mixture was thenpanned on immunotubes coated with either BSA–digoxin or with Protective Antigen protein (PA). Afterwashing, phages that remained bound were eluted viainfection of E. coli Y1088 cells. Infected cells wereplated and the DNA inserts ( pRheo or bla) from thephage genome of well-separated plaques were ampli-fied by PCR and analyzed by agarose gel electropho-resis. As expected, only phage harboring bla wereeluted from the immunotube coated with BSA–di-goxin (Fig. 4a). However, a single round of panningon PA resulted in a 30-fold enrichment of λgt11-

Fig. 4. Enrichment of λ phage displaying the PA-specific 1Hhum scFv.λgt11-pRheo–1Hhum scFv–gpD (108 pfu) were mixed with an excessof λgt11-bla–26.10 scFv–gpD (4×109 pfu). Using primer pairs forboth pRheo and bla, DNA was amplified by PCR from isolatedplaques following panning on: BSA–digoxin (a) or PA (b). In bothexperiments only 12 of the 15 PCR reactions gave a detectable product.(a) All plaques contained the 1.6 kb bla insert which is present inλgt11-bla–26.10 scFv–gpD, (b) 8 out of 12 plaques contained the2.8 kb pRheo insert from λgt11-pRheo–1Hhum scFv–gpD.

Fig. 6. ELISA analysis of phage isolated after two rounds of screening.Binding of identical titers of recombinant λgt11–26.10 scFv–gpD, con-taining E. coli genomic fragments (dark grey columns) or control phage,λgt11-pRheo–26.10 scFv–gpD, containing an unrelated insert (whitecolumns) to BSA–digoxin was monitored by ELISA. Phage prior topanning on BSA–digoxin-coated immunotubes (lanes 3,4) and fol-lowing two rounds of panning on BSA–digoxin-coated immunotubes(lanes 1,2). Bound phages were detected by rabbit polyclonal anti-λ anti-sera followed by goat-anti-rabbit-IgG (H+L)–HRP conjugate.

Fig. 5. Schematic diagram of the λ phage selection strategy for the isolation of genes that affect scFv–gpD display in trans.

170 R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

pRheo–1Hhum scFv–gpD from a phage populationoriginally dominated by λgt11-bla–26.10 scFv–gpD.Eight of the 12 PCR products obtained correspondedto pRheo (Fig. 4b).

3.3. Screening of a genomic library for enhanceddisplay of 26.10 scFv

Panning was used to enrich for clones of chromo-somal DNA that improved the yield of 26.10 scFv–gpD(Fig. 5). A library of λgt11 containing cloned BL21DNA was used to lysogenize E. coli DR473H (pAR-26.10 scFv–gpD). A pool of lysogens was induced withIPTG, and also with arabinose to initiate, respectively,expression of the cloned chromosomal DNA and of26.10 scFv–gpD. Lytic phage growth was then initiatedand the resulting phage was subjected to two rounds ofpanning on immunotubes coated with BSA–digoxinwith bound phage eluted by infection of Y1088. Asignificant increase in the ELISA signal with BSA–digoxin was obtained for phage encoding genomic DNAfragments (Fig. 6).

Thirty individual phages recovered after the secondround were amplified and the one exhibiting thehighest ELISA signal was selected for further study.The cloned BL21 DNA in λgt11 was sequenced,

revealing the promoter and the N-terminal residues(1–235) of the RNA polymerase α-subunit (α-NTD).The cloned region includes the complete N-terminal

171R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

domain of α-NTD (Lloyd et al., 2002). In order toevaluate the effect of the cloned α-NTD on the yieldof soluble proteins E. coli DR473H (λgt11-α-NTD)was transformed with pAR-26.10 scFv–gpD fusion orwith pAR-26.10 scFv. Expression of α-NTD wasinduced with IPTG and then of scFv or scFv–gpDfusion with arabinose. After 2.5 h of growth in ara-binose, cells were lysed and antibody levels deter-mined by ELISA (Fig. 7). Expression of α-NTDresulted in a 2-fold improvement of soluble expres-sion of both the fusion protein 26.10 scFv–gpD andthe 26.10 scFv, suggesting that the strategy outlinedin Fig. 5 is suitable for the isolation of genes thatenhance cytoplasmic expression of desired proteins.

Fig. 7. Effect of the α-NTD gene on the expression of scFv and scFv–gpD fusion. The amount of protein was detected by ELISA. E. coliDR473H (pAR-26.10 scFv–gpD) (a) and E. coli DR473H (pAR-26.10) (b) cells lysogenized with λgt11 encoding the α-NTD gene(dark grey columns) or control λgt11-pRheo (white columns). Datarepresent the average of two measurements with cell lysates from twoindependent cultures.

4. Discussion

Display technologies have proven exceptionallyuseful for protein engineering applications includingthe isolation of antibodies from synthetic or naïve reper-toire libraries, affinity maturation, detection of protein:protein interactions and the screening of cDNA libraries(Li, 2000). Protein export across the cytoplasmic mem-brane is a prerequisite for most in vivo display methods,including M13, yeast, or bacterial surface display. Thisrequirement for protein secretion imposes several con-straints on the properties of the displayed polypeptide.Proteins that contain positively charged N-termini, foldtoo rapidly, or require the action of specializedchaperones or cofactors, can block secretion and as aresult are displayed poorly or not at all. These problemscan be circumvented by using T7 or λ display, becausefusions to their surface proteins assemble entirely in thecytoplasm (Danner and Belasco, 2001; Sokoloff et al.,2000; Yamamoto et al., 1999). Phage λ has provenparticularly useful for display and screening of peptides,complex multimeric proteins and cDNA libraries. Themost permissive display mode is through fusion to theN- or C-terminus of the gpD protein. Multiple copies ofthe fusion protein are incorporated into the virion.

Two important limitations of λ display are the in-efficiency of cloning and library generation and theinability to express proteins containing disulfide bondswithin the reducing cytoplasm of E. coli. Cloning into λand packaging the DNA is less efficient than cloning intoplasmids or phagemids. However, the Cre–Lox systemhas been used to efficiently transfer genes encoding gpDfusions from a plasmid library onto infecting λ phage byrecombination in vivo (Gupta et al., 2003). A scFv spe-cific for the cancer antigen mesothelin was successfullydisplayed on λ when expressed as a gpD fusion in wild-type E. coli. In that instance the disulfide bonds that arecritical for the stability of the VH and VL domains in thescFv were concluded to have formed by oxidation aftercell lysis (Gupta et al., 2003). However, since mostantibody fragments, including the 26.10 scFv, are proneto aggregation in the absence of disulfide bonds they arenot active following expression in the cytoplasm of wild-type E. coli (der Maur et al., 2002; Levy et al., 2001;Ramm et al., 1999).

We overcame the problem of aggregation in bacterialcell cytoplasm by using a specially designed E. colistrain. E. coli trxB gor has a cytoplasmic redox potentialthat approximates that of the eukaryotic endoplasmicreticulum and thus results in efficient protein oxidationfor many multi-disulfide proteins (Ritz and Beckwith,2001). The cytoplasmic environment of these cells

172 R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

allows the expression of functional 26.10 and anti-PAscFv and, following fusion to gpD, their efficient displayon λ. Importantly, antibodies displayed on λ can beenriched by panning on immobilized antigen from alarge excess of phage displaying an antibody of unrelatedspecificity.

In developing this system, scFv–gpD fusions wereexpressed from a low copy number plasmid while wild-type gpD was provided by λ, leading to competition forthe amount of 26.10 scFv–gpD displayed. If necessary,higher display levels could be obtained using highercopy number plasmids, stronger promoters, or moreefficient translation initiation regions (Olins et al.,1988); alternatively, a D amber or other conditionalmutant of λ could be used.

We were interested in isolating genes whose over-expression enhances display and soluble expression ofscFvs by acting in trans to assist folding. Because suchgenes cannot be predicted a priori, we combined thecloning vector properties of λ with its display capacity.The key premise of our approach is that cells over-expressing appropriate genes that assist scFv–gpDfolding will give rise to λ particles that better displaythe fusion protein. These phage particles will in turnexhibit stronger binding onto an antigen by virtue ofimproved display. A similar approach was used in M13display to isolate genes that enhance the folding ofantibody fragments, and to isolate improved disulfideisomerases (Bothmann and Pluckthun, 1998, 2000,Lafond et al., 2003). A selection for trans-acting genesusing λ is more demanding as it requires sequentialinduction of protein expression from the cloned genefragments and then from the plasmid-born scFv–gpDfusion, and also switching from lysogenic to lyticgrowth. We have introduced a key improvement byusing an hfl (high frequency of lysogenization) mutantof E. coli that greatly simplifies library construction.The ΔhflKC::Km mutation increases the stability of theλ cII protein, resulting in a lysogenization efficiency ofabout 90%. In this manner, a clear enhancement ofantigen binding activity was detected in phage isolatedafter only two rounds of panning (Fig. 6).

Interestingly, the N-terminal domain of the E. coliRNA polymerase (α-NTD) was isolated in this screen asa gene product that improved display efficiency.Expression of α-NTD from the λgt11 prophage in-creased soluble levels of both the 26.10 scFv and the26.10 scFv–gpD fusion. The α-NTD protein wasexpressed from the lac promoter in λgt11 and ac-cumulated at moderate levels within the cytoplasm.Higher expression of α-NTD from multicopy plasmidsdid not lead to any additional increase in the soluble

levels of the antibody fragment or the gpD fusion (datanot shown). The N-terminal and C-terminal domains ofthe α-subunit of RNA polymerase fold independentlyand are connected by a flexible linker (Blatter et al.,1994). The α-NTD domain is crucial both for dimer-ization of α, and for the α dimer to interact with theremaining subunits of RNA polymerase, and also forbasal transcription (Ebright and Busby, 1995). Themechanism by which α-NTD is able to facilitate 26.10scFv folding is not clear. α-NTD may have a directeffect on 26.10 scFv synthesis and folding or it may altercellular chaperones in subtle ways that indirectly resultin optimal folding. We note that about 30% of the 26.10scFv normally accumulates in soluble form in the cyto-plasm of mutant cells. Screening for improved displayof less soluble antibody fragments, such as the 4–4-20anti-fluorescein scFv used earlier by Bothmann andPluckthun may be more likely to yield chaperone genesthat are directly involved in protein folding.

Asλ is useful both as a cloning and as a display vector,it may be employed for sequential screens for display andfor enhanced folding. Furthermore, this approach does notrequire prior knowledge of what chaperones or otherproteins may do to improve yields of soluble proteins.

Acknowledgements

We would like to thank Drs. Adhya, Kihara, Boydand Beckwith for providing anti-λ anti-sera, hflA, andtrxB gor mutant E. coli strains, respectively. This workwas supported by grants from the Beckman ResearchTechnology Initiative Award (Project # 26750695) toGG & BLI and NIH # R01 GM55090 to GG.

References

Ansuini, H., Cicchini, C., Nicosia, A., Tripodi, M., Cortese, R.,Luzzago, A., 2002. Nucleic Acids Res. 30, e78.

Beghetto, E., Pucci, A.,Minenkova, O., Spadoni, A., Bruno, L., Buffolano,W., Soldati, D., Felici, F., Gargano, N., 2001. Int. J. Parasitol. 31, 1659.

Bessette, P.H., Aslund, F., Beckwith, J., Georgiou, G., 1999. Proc. Natl.Acad. Sci. U. S. A. 96, 13703.

Blatter, E.E., Ross, W., Tang, H., Gourse, R.L., Ebright, R.H., 1994.Cell 78, 889.

Bothmann, H., Pluckthun, A., 1998. Nat. Biotechnol. 16, 376.Bothmann, H., Pluckthun, A., 2000. J. Biol. Chem. 275, 17100.Boyd, D.,Weiss, D.S., Chen, J.C., Beckwith, J., 2000. J. Bacteriol. 182,

842.Burks, E.A., Chen,G., Georgiou, G., Iverson, B.L., 1997. Proc. Natl. Acad.

Sci. U. S. A. 94, 412.Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias,

M., Velligan, M.D., Wong, W.L., Rowland, A.M., Kotts, C.E.,Carver, M.E., et al., 1992. Biotechnology (N Y) 10, 163.

Chen, G., Dubrawsky, I., Mendez, P., Georgiou, G., Iverson, B.L.,1999. Protein Eng. 12, 349.

173R. Levy et al. / Journal of Immunological Methods 321 (2007) 164–173

Cicchini, C., Ansuini, H., Amicone, L., Alonzi, T., Nicosia, A., Cortese,R., Tripodi, M., Luzzago, A., 2002. J. Mol. Biol. 322, 697.

Danner, S., Belasco, J.G., 2001. Proc. Natl. Acad. Sci. U. S. A. 98,12954.

der Maur, A.A., Zahnd, C., Fischer, F., Spinelli, S., Honegger, A.,Cambillau, C., Escher, D., Pluckthun, A., Barberis, A., 2002.J. Biol. Chem. 277, 45075.

Derman, A.I., Prinz, W.A., Belin, D., Beckwith, J., 1993. Science 262,1744.

Dunn, I.S., 1995. J. Mol. Biol. 248, 497.Ebright, R.H., Busby, S., 1995. Curr. Opin. Genet. Dev. 5, 197.Forrer, P., Jaussi, R., 1998. Gene 224, 45.Francisco, J.A., Campbell, R., Iverson, B.L., Georgiou, G., 1993. Proc.

Natl. Acad. Sci. U. S. A. 90, 10444.Gupta, A., Onda,M., Pastan, I., Adhya, S., Chaudhary, V.K., 2003. J.Mol.

Biol. 334, 241.Guzman, L.M., Belin, D., Carson, M.J., Beckwith, J., 1995. J. Bacteriol.

177, 4121.Hagiwara, H., Kunihiro, S., Nakajima, K., Sano, M., Masaki, H.,

Yamamoto, M., Pak, J.W., Zhang, Y., Takase, K., Kuwabara, I.,Maruyama, I.N., Machida, M., 2002. J. Biochem. (Tokyo) 132,975.

Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L.,Rogers, G.K., 2004. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.

Hayhurst, A., Harris, W.J., 1999. Protein Expr. Purif. 15, 336.Hayhurst, A., Happe, S., Mabry, R., Koch, Z., Iverson, B.L., Georgiou,

G., 2003. J. Immunol. Methods 276, 185.Heo, M.A., Kim, S.H., Kim, S.Y., Kim, Y.J., Chung, J., Oh, M.K., Lee,

S.G., 2005. Protein Expr. Purif.Hermanson, G.T., 1996. Bioconjugate Techniques. Academic Press

Inc, San Diego, CA.Hoess, R.H., 2002. Curr. Pharm. Biotechnol. 3, 23.Horn,U., Strittmatter,W.,Krebber,A.,Knupfer,U., Kujau,M.,Wenderoth,

R.,Muller, K.,Matzku, S., Pluckthun,A., Riesenberg, D., 1996. Appl.Microbiol. Biotechnol. 46, 524.

Huynh, 1985. DNA Cloning, vol. 1. IRL Press Ltd., Oxford, England.Jurado, P., de Lorenzo, V., Fernandez, L.A., 2006. J. Mol. Biol. 357, 49.Kihara, A., Akiyama, Y., Ito, K., 1996. EMBO J. 15, 6122.Lafond, R., Zhan, X., Georgiou, G., 2003. Methods Mol. Biol. 230,

239.Levy, R., Weiss, R., Chen, G., Iverson, B.L., Georgiou, G., 2001.

Protein Expr. Purif. 23, 338.Li, M., 2000. Nat. Biotechnol. 18, 1251.Lloyd, G.S., Niu, W., Tebbutt, J., Ebright, R.H., Busby, S.J., 2002.

Genes Dev. 16, 2557.

Maruyama, I.N., Maruyama, H.I., Brenner, S., 1994. Proc. Natl. Acad.Sci. U. S. A. 91, 8273.

Mikawa, Y.G., Maruyama, I.N., Brenner, S., 1996. J. Mol. Biol. 262,21.

Monje-Casas, F., Jurado, J., Prieto-Alamo, M.J., Holmgren, A., Pueyo,C., 2001. J. Biol. Chem. 276, 18031.

Moriki, T., Kuwabara, I., Liu, F.T., Maruyama, I.N., 1999. Biochem.Biophys. Res. Commun. 265, 291.

Olins, P.O., Devine, C.S., Rangwala, S.H., Kavka, K.S., 1988. Gene73, 227.

Panka, D.J., Mudgett-Hunter, M., Parks, D.R., Peterson, L.L.,Herzenberg, L.A., Haber, E., Margolies, M.N., 1988. Proc. Natl.Acad. Sci. U. S. A. 85, 3080.

Perez-Perez, J., Gutierrez, J., 1995. Gene 158, 141.Philibert, P., Martineau, P., 2004. Microb. Cell Fact. 3, 16.Ramm, K., Gehrig, P., Pluckthun, A., 1999. J. Mol. Biol. 290, 535.Ritz, D., Beckwith, J., 2001. Annu. Rev. Microbiol. 55, 21.Ritz, D., Beckwith, J., 2002. Methods Enzymol. 347, 360.Santi, E., Capone, S., Mennuni, C., Lahm, A., Tramontano, A.,

Luzzago, A., Nicosia, A., 2000. J. Mol. Biol. 296, 497.Santini, C., Brennan, D., Mennuni, C., Hoess, R.H., Nicosia, A.,

Cortese, R., Luzzago, A., 1998. J. Mol. Biol. 282, 125.Segatori, L., Murphy, L., Arredondo, S., Kadokura, H., Gilbert, H.,

Beckwith, J., Georgiou, G., 2006. J. Biol. Chem. 281, 4911.Slominska, M., Neubauer, P., Wegrzyn, G., 1999. Virology 262, 431.Smith, T.W., Butler Jr., V.P., Haber, E., 1970. Biochemistry 9, 331.Sokoloff, A.V., Bock, I., Zhang, G., Sebestyen, M.G., Wolff, J.A.,

2000. Molec. Ther. 2, 131.Sternberg, N., Hoess, R.H., 1995. Proc.Natl. Acad. Sci. U. S. A. 92, 1609.Stolz, J., Ludwig, A., Sauer, N., 1998. FEBS Lett. 440, 213.Studier, F.W., Rosenberg, A.H., Dunn, J.J., Dubendorff, J.W., 1990.

Methods Enzymol. 185, 60.Worn, A., Pluckthun, A., 2001. J. Mol. Biol. 305, 989.Wu, R., Taylor, E., 1971. J. Mol. Biol. 57, 491.Yamamoto, M., Kominato, Y., Yamamoto, F., 1999. Biochem.

Biophys. Res. Commun. 255, 194.Yang, F., Forrer, P., Dauter, Z., Conway, J.F., Cheng, N., Cerritelli, M.E.,

Steven, A.C., Pluckthun, A., Wlodawer, A., 2000. Nat. Struct. Biol.7, 230.

Young, R.A., Davis, R.W., 1983. Proc. Natl. Acad. Sci. U. S. A. 80,1194.

Zucconi, A., Dente, L., Santonico, E., Castagnoli, L., Cesareni, G.,2001. J. Mol. Biol. 307, 1329.