Protein Domain Mapping by λ Phage Display: The Minimal Lactose-Binding Domain of Galectin-3

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<ul><li><p>Protei laLactos</p><p>Takanor chDepartme eyand *Alle 10San Diego</p><p>Received S</p><p>Mappintion isstructurephage ldeterminof humandigestedThe libration usinsequencethe minitose binddues. Usmined rbetweenthe phageither pul surfacedomain mromolecu</p><p>Manythat aretions sucride. Main many biological phenomena such as signal proteiof biologiThis has</p><p>ofnsentan</p><p>inal</p><p>hyffimcu</p><p>mep</p><p>umlibxteed</p><p>se4in</p><p>ctiexphaslMrotpr</p><p>We [10, 11] and others [12, 13] have constructed sur-</p><p>Abbreviaphage plaq</p><p>1 Presentand Immun92121.</p><p>2 To whoCell BiologNorth Torr784-9740. E</p><p>Biochemical and Biophysical Research Communications 265, 291296 (1999)</p><p>Article ID bbrc.1999.1666, available online at onTherefore, determination of such domainsn molecules is essential for an understandingcal and biochemical functions of the protein.been frequently achieved by dissecting cDNA</p><p>face display vectors based on bacteriophage l since thephage particle assembles in the bacterial cytoplasm.These vectors have been used to express both cyto-plasmic and secreted proteins on the surface of thevector phage.</p><p>Gal-3 is a member of an animal lectin family thatbinds polysaccharides containing a b-galactoside moi-ety, which has been shown to have a variety of biolog-ical functions [14]. Among the family, Gal-3 has aunique structure in which its amino-terminal regioncontains proline and glycine-rich tandem repeats and</p><p>tions used: Gal-3, human lectin galectin-3; pfu, bacterio-ue-forming unit.address: Allergy Division, La Jolla Institute for Allergyology, 10355 Science Center Dr., San Diego, California</p><p>m correspondence should be addressed at Department ofy, Mail Drop MB-30, Scripps Research Institute, 10550n Domain Mapping by l Phage Dispe-Binding Domain of Galectin-3</p><p>i Moriki, Ichiro Kuwabara,1 Fu-Tong Liu,* and Int of Cell Biology, Scripps Research Institute, 10550 N. Torrrgy Division, La Jolla Institute for Allergy and Immunology,, California 92121</p><p>eptember 15, 1999</p><p>g of protein domains having a distinct func-essential to understanding the proteinsfunction relationship. We used a bacterio-surface expression vector, lfoo, in order toe the minimal carbohydrate-binding domaingalectin-3 (Gal-3). Gal-3 cDNA was randomly</p><p>by DNase I and cloned into the phage vector.ry generated was screened by affinity selec-g lactose immobilized on agarose beads. DNA</p><p>analysis of a set of isolated clones definedmal folding domain of Gal-3 required for lac-ing, which consisted of 136 amino-acid resi-</p><p>ing the phage clones isolated, we also deter-elative dissociation constants in solutionlactose and the minimal domain expressed one surface. This technique does not requirerified or labeled proteins, and bacteriophagedisplay may, therefore, be useful for proteinapping and in vitro studies of various mac-</p><p>lar interactions. 1999 Academic Press</p><p>proteins have multiple segments or domainsinvolved in distinct macromolecular interac-h as protein-protein and protein-polysaccha-cromolecular interactions play central roles</p><p>clonesPortiofragmlines,vivo oring anyeastlate suof theromole</p><p>Filaisolatevast ncDNAhave e[reviewexpresin ref.determinterabrarytous pto tranbrane.coat pfusionis involvterminal</p><p>ey Pines Road, La Jolla, California 92037. Fax: (858)-mail:</p><p>291y: The Minimal</p><p>i N. Maruyama2</p><p>Pines Road, La Jolla, California 92037;355 Science Center Drive,</p><p>interest with restriction enzymes or DNase I.of the protein encoded by the dissected DNAs are expressed in bacteria or cultured celld subsequently examined for their activity in</p><p>vitro. However, existing approaches, includ-yses of phage plaques, bacterial colonies orbrid systems, are not efficient enough to iso-cient numbers of clones for the determinationinimal folding domains required for the mac-lar interactions.ntous fusion phage is a powerful means to</p><p>articular clones from a library consisting of aber of variants, such as random peptide orraries [1, 2]. The random peptide librariesnsively been used for B cell epitope mappingin ref. 3], and various proteins have been</p><p>d on the surface of the vector phage [reviewed]. The phage display has also been used toe small peptides required for macromolecularons by the affinity selection of the phage li-ressing protein fragments [57]. The filamen-</p><p>ge vectors rely on the ability of fusion proteinsocate across the bacterial cytoplasmic mem-any water-soluble proteins fused to the phageeins may interfere with the passage of theoduct from the cytoplasm to periplasm [8, 9].ed in self-association [15]. The carboxyl-half of Gal-3 is responsible for the carbohy-</p><p>0006-291X/99 $30.00Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.</p></li><li><p>drate recognition in common to other galectin familymembers. Gal-3 interacts with extracellular glycocon-jugates such as IgE [16, 17], laminin [18], and mucin[19] as wcytosol [2the dissoM depenIt is impohydrateterminalof the stand for ting mutaterminatresponsibtion of rwith thethe outsilactose adetermin</p><p>MATERIA</p><p>Library cII SK(2) (Srandom frabrief, apprwith DNasbp and 200and long-inby T4 DNAligated witgated adapfragments,lfooDc DNaging the liby infectinlong-insertand 4 3 10</p><p>A long-inGal-3 carboGal-3 39-terwas amplifiGal-3 cDNAGCGCCCCATGGTATAEcoRI andused for thsisted of 4 3</p><p>Affinity sstrain TG1surface of tphages we8000, Fishein 0.5 mlsodium azi0.45 ml, waLouis, MO;ml) and incentrifugatfuge, and10-min milthen three</p><p>beads was eluted with 0.2 ml of l-dil containing 20 mM lactose. Halfof the eluate was used for phage titration and the other half for thesecond selection as above. After three rounds of affinity selection,</p><p>laqupophe. Al off blnge mngph</p><p>thetheactlaqufa</p><p>catas enc</p><p>d inrevthd cs wmfra</p><p>witteisayncoamheeci</p><p>nobby iage</p><p>ferbuf1%</p><p>in.lect</p><p>elwi</p><p>Cl,-3se-gy,am,</p><p>ciatereoftraagdil0.5</p><p>r thfte</p><p>gatithaClmlph</p><p>inpof</p><p>eme</p><p>Vol. 265, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSell as with RNA both in the nucleus and0, 21] It has a specific affinity for lactose withciation constants that range from 1023 to 1025</p><p>ding on experimental conditions used [15, 22].rtant to know the minimal domain for carbo-binding by separating it from the amino-self-association domain for an understandingructure-function relationship of the protein,he subsequent analysis of the domain includ-genesis. In this report, we describe the de-</p><p>ion of the minimal folding domain of Gal-3le for lactose binding by lactose-affinity selec-</p><p>andom Gal-3 fragment libraries constructedl phage vector lfoo; foo stands for fusion onde. The relative dissociation constant betweennd the minimal domain of Gal-3 was alsoed by this technology.</p><p>LS AND METHODS</p><p>onstruction. Gal-3 cDNA, 914 bp, cloned in pBluescripttratagene, La Jolla, CA) was used for the construction ofgment libraries by methods previously described [23]. Inoximately 10 mg of this plasmid construct was digestede I, fractionated by agarose gel, and two fractions, 70120500 bp, were purified for the construction of short-insertsert libraries, respectively. The fragments were bluntedpolymerase, and then 0.4 mg of the resultant DNA was</p><p>h 20-fold molar excess of adaptors. After removing unli-tors by agarose gel electrophoresis, the purified DNAapproximately 30 ng, were ligated with 1.0 mg of the</p><p>A [ref. 11] digested with BamHI and EcoRI. After pack-gation mixtures, resulting phage libraries were amplifiedg an Escherichia coli strain, Q526. The short-insert andlibraries comprised of 1.2 3 106 plaque forming unit (pfu)6 pfu independent recombinant clones, respectively.sert library was also made from a plasmid containing thexyl-terminal half, using the same procedures above. Theminal half, ranging from the nucleotide number 313 to 750,ed by the polymerase chain reaction (PCR) [24] using the</p><p>as template and a pair of primers, 59-TTAAGCTTTG-TGCTGGGCCACTG and 59-GCCGAATTCTCATTATATC-TGAAGC. The PCR product was digested with HindIII and</p><p>cloned into pBluescript II SK(2). This plasmid was directlye library construction as described above. The library con-</p><p>105 independent recombinant clones.</p><p>election of libraries. Libraries were grown with E. coliin 5.0 ml CY medium to produce fusion proteins on thehe phage particle as described [11]. After complete lysis,re precipitated by adding 5% polyethylene glycol (PEGr) and 0.5 M NaCl at final concentrations, and suspended</p><p>of blocking buffer [1% BSA, 0.1% Tween 20, and 0.1%de in phosphate buffered saline (PBS)]. The suspension,s mixed with 50 ml of lactosyl agarose beads (Sigma, St.the binding capacity of 24 mg Arachis hypogea lectin percubated overnight at 4C. The beads were collected byion at 14,000 rpm for a few seconds in Eppendorf centri-washed three times with 1.0 ml of blocking buffer byd shaking at room temperature between the washing andtimes with 1.0 ml of l-dil [ref. 10]. Phage bound to the</p><p>white-plibraryeach of t2 ml CYin 0.1 m0.1 ml ocontainiGal). Thin bindiforming95% ofwere furbindingphage pthe man</p><p>PurifiGal-3 w[15]. Truproduceity as pMet-130PCR anplasmidtransforof Gal-3lysatession prowere asstruct eenoughassay wusing sp</p><p>Immutivatedlysis, phTM bufsampleglycerol,for 10 m(10%) eusing anblockingmM Naanti-GalperoxidatechnoloAmersh</p><p>DissoMC8 wvolumesa concenpfu of phseriallybeads inused fobelow. Acentrifutimes w0.2 M Nwith 20releasedratio ofnumbermeasur</p><p>292e forming phages were enriched to more than 80% of theulation. These white plaques were randomly picked andphage clones was separately amplified by infecting TG1 infter precipitation with PEG, the individual clones, 107 pfublocking buffer, were mixed with vector phage, 108 pfu in</p><p>ocking buffer, that can form a blue plaque on an agar plate25 mg/ml 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-ixture was incubated with 10 ml of lactosyl agarose beadsbuffer, washed, and eluted as above. The white-plaqueages were greatly enriched over blue vectors, more thanmixture, after a single affinity selection. These phagesr analyzed by DNA sequencing as clones having a lactose-ivity. DNA sequencing was directly carried out from theues, using a Cyclist Exo-Pfu sequencing kit according tocturers protocol (Stratagene).</p><p>ion of Gal-3 and its truncated form. The full length ofxpressed in bacteria and purified as previously described</p><p>ated forms of the carboxyl-terminal half of Gal-3 were alsobacteria in order to examine their lactose-binding activ-</p><p>iously described [25]. Briefly, a DNA fragment encodingrough Ile-250 or Gly-112 through Ile-240 was amplified byloned into pGEX-5X-3 (Pharmacia), and the resultingere confirmed by DNA sequencing and were then used toE. coli. Glutathione-S-transferase (GST) fusion productsgments were isolated by affinity purification of bacterialh glutathione agarose. After removing GST from the fu-ns by factor Xa cleavage, the truncated forms of Gal-3ed for lactose binding as described [26]. The latter con-ding from Gly-112 to Ile-240 of Gal-3 failed to express theount of the GST fusion product for the lactose-binding</p><p>n analyzed by Western blotting of the bacterial lysatefic antibodies as probes.</p><p>lotting of phage fusion proteins. Phage clones were cul-nfecting TG1 or MC8 [ref. 10] in 50 ml CY. After completes were precipitated twice with PEG, resuspended in 50 ml(10 mM TrisHCl, pH 8.0, 10 mM MgCl2), and boiled infer [62.5 mM TrisHCl, pH 6.8, 2% (w/v) SDS, 10% (v/v)</p><p>(v/v) 2-mercaptoethanol, 0.005% (w/v) bromophenol blue]Phage proteins were separated by SDSpolyacrylamide gelrophoresis, and then transferred to a nitrocellulose filterectroblotting apparatus (Bio-Rad, Hercules, CA). After pre-th Blotto (5% nonfat dry milk, 10 mM TrisHCl, pH 8.0, 1500.05% Tween 20), the membrane was immunostained withmonoclonal antibody, 1H11 [ref. 23], and horseradishconjugated goat anti-mouse IgG antibody (Santa Cruz Bio-Santa Cruz, CA), using a color development kit (ECL;Little Chalfont, England).</p><p>ion constant measurement. Phage clones grown withprecipitated by PEG and resuspended in appropriate</p><p>binding buffer (0.1% NaN3, 0.1% Tween 20 in PBS) to givetion of approximately 1010 pfu/ml. To estimate the total</p><p>e bound to lactose beads, half of the phage suspension wasuted with binding buffer and mixed with 10 ml of lactosyl</p><p>ml of binding buffer; the other half of the suspension wase measurement of dissociation constants as describedr incubation for 4 h at 4C, the beads were collected byion, washed three times with binding buffer, and threewashing buffer (10 mM TrisHCl, pH 7.4, 5 mM MgCl2,). Bound phages were eluted from the beads by incubatingof l-dil containing 20 mM lactose. After titration of theage by plating with a bacterial host, JM105 [ref. 27], the</p><p>ut phage titer to output was used for the estimation of thephage that had an ability to bind lactose in subsequentnt of relative dissociation constants.</p></li><li><p>For the measurement of relative dissociation constants betweenphage clones and lactose in solution, the other half of the phagesuspension from the above preparation was mixed with variousconcentratibinding bubeads waswas furthecentrifugattrated to coThe total pfrom the litained abofragments,tion was calactose-unbalso assumphage andby the addof lactose-uless than 0tween phaScatchard</p><p>RESULTS</p><p>Affinityfine thelactose bshort or linto twotively, bphages wcoli straifusion prThe lfooafter thethe Gal-3duction osuppresspressedbeads tophage poselectioning phagmore thaphage cldominatetor phagplate consuch a whall fromgesting trangingtivity for</p><p>To exalactose, wlong-inseformingproximatwhite-plalection o</p><p>folaqth</p><p>ong cedhe</p><p>pintlenayl-etenecincarag[15ethutagd</p><p>ncoof</p><p>ialreseocetinsefor</p><p>1.selD</p><p>39-etermn wt nubef-receatbe</p><p>hag</p><p>Vol. 265, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSons of lactose, ranging from 0.25 to 5 mM, in 0.5 ml offfer. After incubation overnight at 4C, 10 ml of lactosyladded to capture lactose-unbound phage, and the mixturer incubated for 4 h at 4C. After collecting the beads byion, phage bound to beads was eluted as above, and ti-unt the number of phage that did not bind free lactose.fu of lactose-unbound phage in solution was estimatednear relationship between input and output phage ob-</p><p>ve. Assuming that phage displays one molecule of Gal-3the concentration of lactose bound to phage in the solu-</p><p>lculated by subtracting the estimated concentration of theound phage from the concentration of input phage. It wased that, when the equilibrium of the reaction betweenlactose was reached, the equilibrium was not influencedition of lactosyl beads into the solution since the fractionnbound phage collected by lactosyl beads is very small,.005% of total phage. Relative dissociation constants be-ge clones and lactose were obtained from the slope ofplots.</p><p>selection of Gal-3 fragment libraries. To de-minimal segment of the Gal-3 molecule forinding, we made two libraries, containingong inserts, from the Gal-3 cDNA fractionatedsize ranges, 70120 bp or 200500 bp, respec-y agarose gel electrophoresis. The libraryere cultivated with a suppressor-positive E.</p><p>n, TG1, to produce Gal-3 random fragments asoteins on the surface of the phage particle.vector ha...</p></li></ul>