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Biochemical and Biophysical Research Communications 265, 291–296 (1999)

Article ID bbrc.1999.1666, available online at http://www.idealibrary.com on

rotein Domain Mapping by l Phage Display: The Minimalactose-Binding Domain of Galectin-3

akanori Moriki, Ichiro Kuwabara,1 Fu-Tong Liu,* and Ichi N. Maruyama2

epartment of Cell Biology, Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037;nd *Allergy Division, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive,an Diego, California 92121

eceived September 15, 1999

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Mapping of protein domains having a distinct func-ion is essential to understanding the protein’structure–function relationship. We used a bacterio-hage l surface expression vector, lfoo, in order toetermine the minimal carbohydrate-binding domainf human galectin-3 (Gal-3). Gal-3 cDNA was randomlyigested by DNase I and cloned into the phage vector.he library generated was screened by affinity selec-

ion using lactose immobilized on agarose beads. DNAequence analysis of a set of isolated clones definedhe minimal folding domain of Gal-3 required for lac-ose binding, which consisted of 136 amino-acid resi-ues. Using the phage clones isolated, we also deter-ined relative dissociation constants in solution

etween lactose and the minimal domain expressed onhe phage surface. This technique does not requireither purified or labeled proteins, and bacteriophagesurface display may, therefore, be useful for proteinomain mapping and in vitro studies of various mac-omolecular interactions. © 1999 Academic Press

Many proteins have multiple segments or domainshat are involved in distinct macromolecular interac-ions such as protein-protein and protein-polysaccha-ide. Macromolecular interactions play central rolesn many biological phenomena such as signal trans-uction. Therefore, determination of such domainsn protein molecules is essential for an understandingf biological and biochemical functions of the protein.his has been frequently achieved by dissecting cDNA

Abbreviations used: Gal-3, human lectin galectin-3; pfu, bacterio-hage plaque-forming unit.

1 Present address: Allergy Division, La Jolla Institute for Allergynd Immunology, 10355 Science Center Dr., San Diego, California2121.

2 To whom correspondence should be addressed at Department ofell Biology, Mail Drop MB-30, Scripps Research Institute, 10550orth Torrey Pines Road, La Jolla, California 92037. Fax: (858)84-9740. E-mail: ichi@scripps.edu.

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lones of interest with restriction enzymes or DNase I.ortions of the protein encoded by the dissected DNA

ragments are expressed in bacteria or cultured cellines, and subsequently examined for their activity inivo or in vitro. However, existing approaches, includ-ng analyses of phage plaques, bacterial colonies oreast hybrid systems, are not efficient enough to iso-ate sufficient numbers of clones for the determinationf the minimal folding domains required for the mac-omolecular interactions.Filamentous fusion phage is a powerful means to

solate particular clones from a library consisting of aast number of variants, such as random peptide orDNA libraries [1, 2]. The random peptide librariesave extensively been used for B cell epitope mappingreviewed in ref. 3], and various proteins have beenxpressed on the surface of the vector phage [reviewedn ref. 4]. The phage display has also been used toetermine small peptides required for macromolecularnteractions by the affinity selection of the phage li-rary expressing protein fragments [5–7]. The filamen-ous phage vectors rely on the ability of fusion proteinso translocate across the bacterial cytoplasmic mem-rane. Many water-soluble proteins fused to the phageoat proteins may interfere with the passage of theusion product from the cytoplasm to periplasm [8, 9].

e [10, 11] and others [12, 13] have constructed sur-ace display vectors based on bacteriophage l since thehage particle assembles in the bacterial cytoplasm.hese vectors have been used to express both cyto-lasmic and secreted proteins on the surface of theector phage.Gal-3 is a member of an animal lectin family that

inds polysaccharides containing a b-galactoside moi-ty, which has been shown to have a variety of biolog-cal functions [14]. Among the family, Gal-3 has anique structure in which its amino-terminal regionontains proline and glycine-rich tandem repeats ands involved in self-association [15]. The carboxyl-erminal half of Gal-3 is responsible for the carbohy-

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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rate recognition in common to other galectin familyembers. Gal-3 interacts with extracellular glycocon-

ugates such as IgE [16, 17], laminin [18], and mucin19] as well as with RNA both in the nucleus andytosol [20, 21] It has a specific affinity for lactose withhe dissociation constants that range from 1023 to 1025

depending on experimental conditions used [15, 22].t is important to know the minimal domain for carbo-ydrate binding by separating it from the amino-erminal self-association domain for an understandingf the structure-function relationship of the protein,nd for the subsequent analysis of the domain includ-ng mutagenesis. In this report, we describe the de-ermination of the minimal folding domain of Gal-3esponsible for lactose binding by lactose-affinity selec-ion of random Gal-3 fragment libraries constructedith the l phage vector lfoo; “foo” stands for fusion on

he outside. The relative dissociation constant betweenactose and the minimal domain of Gal-3 was alsoetermined by this technology.

ATERIALS AND METHODS

Library construction. Gal-3 cDNA, 914 bp, cloned in pBluescriptI SK(2) (Stratagene, La Jolla, CA) was used for the construction ofandom fragment libraries by methods previously described [23]. Inrief, approximately 10 mg of this plasmid construct was digestedith DNase I, fractionated by agarose gel, and two fractions, 70–120p and 200–500 bp, were purified for the construction of short-insertnd long-insert libraries, respectively. The fragments were bluntedy T4 DNA polymerase, and then 0.4 mg of the resultant DNA wasigated with 20-fold molar excess of adaptors. After removing unli-ated adaptors by agarose gel electrophoresis, the purified DNAragments, approximately 30 ng, were ligated with 1.0 mg of thefooDc DNA [ref. 11] digested with BamHI and EcoRI. After pack-ging the ligation mixtures, resulting phage libraries were amplifiedy infecting an Escherichia coli strain, Q526. The short-insert andong-insert libraries comprised of 1.2 3 106 plaque forming unit (pfu)nd 4 3 106 pfu independent recombinant clones, respectively.A long-insert library was also made from a plasmid containing theal-3 carboxyl-terminal half, using the same procedures above. Theal-3 39-terminal half, ranging from the nucleotide number 313 to 750,as amplified by the polymerase chain reaction (PCR) [24] using theal-3 cDNA as template and a pair of primers, 59-TTAAGCTTTG-CGCCCCTGCTGGGCCACTG and 59-GCCGAATTCTCATTATATC-TGGTATATGAAGC. The PCR product was digested with HindIII andcoRI and cloned into pBluescript II SK(2). This plasmid was directlysed for the library construction as described above. The library con-isted of 4 3 105 independent recombinant clones.

Affinity selection of libraries. Libraries were grown with E. colitrain TG1 in 5.0 ml CY medium to produce fusion proteins on theurface of the phage particle as described [11]. After complete lysis,hages were precipitated by adding 5% polyethylene glycol (PEG000, Fisher) and 0.5 M NaCl at final concentrations, and suspendedn 0.5 ml of blocking buffer [1% BSA, 0.1% Tween 20, and 0.1%odium azide in phosphate buffered saline (PBS)]. The suspension,.45 ml, was mixed with 50 ml of lactosyl agarose beads (Sigma, St.ouis, MO; the binding capacity of 24 mg Arachis hypogea lectin perl) and incubated overnight at 4°C. The beads were collected by

entrifugation at 14,000 rpm for a few seconds in Eppendorf centri-uge, and washed three times with 1.0 ml of blocking buffer by0-min mild shaking at room temperature between the washing andhen three times with 1.0 ml of l-dil [ref. 10]. Phage bound to the

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f the eluate was used for phage titration and the other half for theecond selection as above. After three rounds of affinity selection,hite-plaque forming phages were enriched to more than 80% of the

ibrary population. These white plaques were randomly picked andach of the phage clones was separately amplified by infecting TG1 inml CY. After precipitation with PEG, the individual clones, 107 pfu

n 0.1 ml of blocking buffer, were mixed with vector phage, 108 pfu in.1 ml of blocking buffer, that can form a blue plaque on an agar plateontaining 25 mg/ml 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-al). The mixture was incubated with 10 ml of lactosyl agarose beads

n binding buffer, washed, and eluted as above. The white-plaqueorming phages were greatly enriched over blue vectors, more than5% of the mixture, after a single affinity selection. These phagesere further analyzed by DNA sequencing as clones having a lactose-inding activity. DNA sequencing was directly carried out from thehage plaques, using a Cyclist Exo-Pfu sequencing kit according tohe manufacturer’s protocol (Stratagene).

Purification of Gal-3 and its truncated form. The full length ofal-3 was expressed in bacteria and purified as previously described

15]. Truncated forms of the carboxyl-terminal half of Gal-3 were alsoroduced in bacteria in order to examine their lactose-binding activ-ty as previously described [25]. Briefly, a DNA fragment encoding

et-130 through Ile-250 or Gly-112 through Ile-240 was amplified byCR and cloned into pGEX-5X-3 (Pharmacia), and the resultinglasmids were confirmed by DNA sequencing and were then used toransform E. coli. Glutathione-S-transferase (GST) fusion productsf Gal-3 fragments were isolated by affinity purification of bacterialysates with glutathione agarose. After removing GST from the fu-ion proteins by factor Xa cleavage, the truncated forms of Gal-3ere assayed for lactose binding as described [26]. The latter con-

truct encoding from Gly-112 to Ile-240 of Gal-3 failed to express thenough amount of the GST fusion product for the lactose-bindingssay when analyzed by Western blotting of the bacterial lysatesing specific antibodies as probes.

Immunoblotting of phage fusion proteins. Phage clones were cul-ivated by infecting TG1 or MC8 [ref. 10] in 50 ml CY. After completeysis, phages were precipitated twice with PEG, resuspended in 50 mlM buffer (10 mM Tris–HCl, pH 8.0, 10 mM MgCl2), and boiled inample buffer [62.5 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v)lycerol, 1% (v/v) 2-mercaptoethanol, 0.005% (w/v) bromophenol blue]or 10 min. Phage proteins were separated by SDS–polyacrylamide gel10%) electrophoresis, and then transferred to a nitrocellulose filtersing an electroblotting apparatus (Bio-Rad, Hercules, CA). After pre-locking with Blotto (5% nonfat dry milk, 10 mM Tris–HCl, pH 8.0, 150M NaCl, 0.05% Tween 20), the membrane was immunostained with

nti-Gal-3 monoclonal antibody, 1H11 [ref. 23], and horseradisheroxidase-conjugated goat anti-mouse IgG antibody (Santa Cruz Bio-echnology, Santa Cruz, CA), using a color development kit (ECL;mersham, Little Chalfont, England).

Dissociation constant measurement. Phage clones grown withC8 were precipitated by PEG and resuspended in appropriate

olumes of binding buffer (0.1% NaN3, 0.1% Tween 20 in PBS) to giveconcentration of approximately 1010 pfu/ml. To estimate the total

fu of phage bound to lactose beads, half of the phage suspension waserially diluted with binding buffer and mixed with 10 ml of lactosyleads in 0.5 ml of binding buffer; the other half of the suspension wassed for the measurement of dissociation constants as describedelow. After incubation for 4 h at 4°C, the beads were collected byentrifugation, washed three times with binding buffer, and threeimes with washing buffer (10 mM Tris–HCl, pH 7.4, 5 mM MgCl2,.2 M NaCl). Bound phages were eluted from the beads by incubatingith 20 ml of l-dil containing 20 mM lactose. After titration of the

eleased phage by plating with a bacterial host, JM105 [ref. 27], theatio of input phage titer to output was used for the estimation of theumber of phage that had an ability to bind lactose in subsequenteasurement of relative dissociation constants.

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hage clones and lactose in solution, the other half of the phageuspension from the above preparation was mixed with variousoncentrations of lactose, ranging from 0.25 to 5 mM, in 0.5 ml ofinding buffer. After incubation overnight at 4°C, 10 ml of lactosyleads was added to capture lactose-unbound phage, and the mixtureas further incubated for 4 h at 4°C. After collecting the beads by

entrifugation, phage bound to beads was eluted as above, and ti-rated to count the number of phage that did not bind free lactose.he total pfu of lactose-unbound phage in solution was estimated

rom the linear relationship between input and output phage ob-ained above. Assuming that phage displays one molecule of Gal-3ragments, the concentration of lactose bound to phage in the solu-ion was calculated by subtracting the estimated concentration of theactose-unbound phage from the concentration of input phage. It waslso assumed that, when the equilibrium of the reaction betweenhage and lactose was reached, the equilibrium was not influencedy the addition of lactosyl beads into the solution since the fractionf lactose-unbound phage collected by lactosyl beads is very small,ess than 0.005% of total phage. Relative dissociation constants be-ween phage clones and lactose were obtained from the slope ofcatchard plots.

ESULTS

Affinity selection of Gal-3 fragment libraries. To de-ne the minimal segment of the Gal-3 molecule for

actose binding, we made two libraries, containinghort or long inserts, from the Gal-3 cDNA fractionatednto two size ranges, 70–120 bp or 200–500 bp, respec-ively, by agarose gel electrophoresis. The libraryhages were cultivated with a suppressor-positive E.oli strain, TG1, to produce Gal-3 random fragments asusion proteins on the surface of the phage particle.he lfoo vector has an amber stop codon immediatelyfter the capsid protein pD-coding region and beforehe Gal-3 fragments. Therefore, the efficiency of pro-uction of the fusion protein is dependent on the amberuppressor activity of bacterial hosts used. These ex-ressed libraries were selected by lactosyl agaroseeads to enrich for lactose-binding clones in the libraryhage population. After three rounds of the affinityelection of the long-insert library, white-plaque form-ng phages were significantly enriched and consisted of

ore than 80% of the population. This indicated thathage clones encoding the lactose-binding domainominated in the library population since the lfoo vec-or phage having no insert formed blue plaques on alate containing the color indicator X-Gal. In contrast,uch a white-plaque forming phage was not enriched atll from the selection of the short-insert library, sug-esting that fragments encoded by the short insertsanging 70–120 bp lack sufficient lactose-binding ac-ivity for the affinity selection.

To examine whether the selected phage could bindactose, we reselected the clones recovered from theong-insert library after mixing with blue plaque-orming vector phages: the mixture consisted of ap-roximately 90% blue-plaque-forming phage and 10%hite-plaque-forming phage. After a single affinity se-

ection of the mixture with lactosyl beads, the white-

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laque forming phage was greatly enriched over thelue-plaque forming phage and consisted of more than5% of the population. This confirmed that the affinityelection of the library indeed enriched for lactose-inding clones. Using the single affinity reselection, wenalyzed 40 white plaque-forming clones recoveredrom the library and isolated 23 lactose-binding clones.

Mapping of lactose-binding domain. To define aegment responsible for lactose binding in the Gal-3olecule, the 23 clones isolated from the libraryere analyzed by DNA sequencing. The amino- and

arboxyl-terminal sequences of the clones, W1–W10,ere determined and are summarized in Fig. 1. Among

he clones, the smallest W10 encoded a small portion ofhe glycine and proline-rich tandem repeats in additiono the carboxyl-terminal half. It has been observed thatal-3 aggregates through self-association of the re-eats [15]. Based on this observation, we suspectedhat the portion of the repeats might increase in affin-ty of the polypeptide for lactosyl beads and mightontribute to more efficient enrichment by facilitatinghage aggregation. To test this hypothesis, we con-tructed a long-insert library from the Gal-3 cDNAhat encodes the carboxyl-terminal half and lacks anyortion of the amino-terminal repeats as described underaterials and Methods. The affinity selection of the

ibrary recovered a number of clones encoding the samensert sequences of which amino termini start at thele-115 of Gal-3 (Fig. 1). All the isolated clones encodedequences beyond the carboxyl-terminal isoleucine,uggesting that all the carboxyl-terminal residues arelso essential for the formation of structural determi-ants for lactose binding. These results indicate that

FIG. 1. DNA sequence analysis of clones isolated by lactose-ffinity selection. Phage clones purified by the selection were ana-yzed by DNA sequencing of the inserts. From the sequences of the9- and 39-ends of the inserts, Gal-3 fragments encoded by the clonesere determined and shown by lines under the Gal-3 cDNA sequencen top, in which its coding region was shown by a rectangle. The firstnd last nucleotides/amino-acid residues encoded by the clone arehown before and after the line, respectively. The Gal-3 carbo-ydrate-recognition domain (CRD) and glycine and proline-rich tan-em repeats are indicated by a filled and hatched box, respectively.he number in parenthesis after the clone name indicates the num-er of phage clones that encode the same insert.

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he Gal-3 domain encompassing Ile-115 through Ile-50 is the minimal folding domain of Gal-3 required foractose binding. This result was also supported by ob-ervations in which respective deletion of 15 or 10mino-acid residues long from the amino- or carboxyl-erminus of the minimal folding domain abolished itsactose binding or stable expression in bacteria, respec-ively (data not shown).

Affinity of fusion phage for lactose. As describedbove, we suspected the glycine and proline-rich tan-em repeats might contribute to the affinity of Gal-3or lactose. We chose three representative clones, C1,

10 and W1, which encode no or varying lengths of thelycine and proline-rich tandem repeats in addition tohe carboxyl-terminal lactose-binding domain. Theselones were grown with an E. coli host, TG1 or MC8, inhich the expression level of the Gal-3 fragments on

he surface of the phage is relatively high or low, re-pectively. We previously measured the expression lev-ls of E. coli b-galactosidase on lfoo and found thatpproximately 35 and 3 molecules of the enzyme perhage particle are incorporated into the phage grownith TG1 and MC8, respectively [11]. As shown in Fig., the levels of incorporation of these Gal-3 fragmentsnto the phage particle grown with TG1 were morehan ten times higher than those with MC8 as ex-ected. From the Western blot analysis of five indepen-ent preparations of the expressed phage grown withG1 or MC8, the average number of the Gal-3 fragmentsisplayed on the phage clones was 90 or 5, respectively.The three phage clones were also used to compare

heir binding rates to lactosyl beads as shown in Fig. 3.wo phage clones, W1 and W10, grown with TG1 were

FIG. 2. Western blot analysis of Gal-3 fragments displayed onhe phage. Phage clones, W1, W10 and C1, were cultivated withither TG1 or MC8, partially purified twice by PEG precipitation,nd solubilized in SDS sample buffer to a concentration of 1012 pfuer ml. Phage proteins equivalent to 109 or 1010 pfu of phage grownith TG1 or MC8, respectively, were separated by SDS–PAGE, blot-

ed onto a nitrocellulose membrane, and stained with anti-Gal-3onoclonal antibody 1H11. The top band in each lane is a fusion

roduct between the phage coat protein pD, ;11 kDa, and Gal-3ragment, and the bottom band represents a cleaved Gal-3 fragment.he vector phage (V) with no insert is also shown as a reference.olecular mass standards (47.5, 32.5, 25, and 16.5 in kilodaltons

rom top) are also indicated by lines on the left.

294

ore efficiently bound to lactosyl beads than C1, buthis higher efficiency was not observed when grownith MC8. This can be explained by that the highxpression level of the Gal-3 fragments promotes theggregation of phage particles through self-associationf the glycine and proline-rich tandem repeats. Theore efficient binding of the W1 and W10 phage grownith TG1 may be accounted for by a “piggy-back” struc-

ure, in which more than one phage particles are boundo a phage particle trapped to lactosyl beads. Indeed,he W1 clone contained a larger portion of the repeatsnd bound more efficiently than W10. The C1 cloneaving none of the repeats bound less efficiently, and

ts binding to the beads was comparable to that of thelones grown with MC8. Dimerization of the lactose-inding domain itself has also been demonstrated26, 28]. This dimerization occurred at concentrationsigher than 0.1 mM of Gal-3 and, therefore, might notontribute significantly in the present phage selection,n which the concentration of Gal-3 displayed on phageas estimated to be less than 0.1 nM. Indeed, the C1hage grown with TG1 or MC8 did not show a signifi-ant difference in binding rates to lactosyl beads.We also used the fusion phage for measuring relative

issociation constants between the phage and free lac-ose molecules in solution. We first defined experimen-al conditions in which a linear relationship betweennput and output phage was observed when the phageas mixed with a constant volume of lactosyl beads forconstant period. As illustrated in Fig. 4A, the linear

elationship was observed when the concentrations ofnput phage ranging from 109 to 1010 pfu were mixedith 10 ml lactosyl beads in the total volume of 0.5 ml

or 4 h at 4°C. Using this standard, we estimated the

FIG. 3. Time course of reaction between isolated phage clonesnd lactosyl beads. The phage clones, W1, W10 and C1, were culti-ated with either TG1 or MC8. Approximately 109 pfu of the phagesere mixed with 10 ml of lactosyl beads in 0.5 ml of blocking buffer.he mixture was incubated for the indicated periods at 4°C, andound phage was eluted with free lactose and counted by plating.he ratios of output phage titer to input are plotted against the

ncubation time; phage clones, W1 (E), W10 (‚) and C1 (h) grownith TG1, and W1 (F), W10 (Œ) and C1 (■) grown with MC8.

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fu of free phage in the reaction mixture containing theal-3 phage and lactose at equilibrium. The three rep-

esentative phage clones, W1, W10 and C1, grown withC8 were separately mixed with various concentra-

ions of free lactose, and the pfu of unbound phage toactose at equilibrium was estimated using the abovetandard after collecting the unbound phage by lacto-yl beads. The number of phage bound to free lactoseas then calculated by subtracting the estimated pfu of

he unbound phage from the total pfu of phage added to

FIG. 4. (A) Linear relationship between input and output phage. Ahage clone, C1, was cultivated with MC8, purified by PEG precipita-ion, and resuspended in binding buffer. 0.1 ml of the suspension,ontaining approximately 1010 pfu of fusion phage, was serially dilutedith binding buffer and then mixed with 10 ml of lactosyl beads in a

otal volume of 0.5 ml binding buffer. The mixture was incubated for 4 ht 4°C and the bound phage was eluted as described under Materialsnd Methods. In this figure, only data from the C1 clone are shown asrepresentative of the three phage clones W1, W10 and C1. Points

epresent the average of three assays and error bars are the standardeviation from the mean. (B) Scatchard analysis for fusion phage boundo lactose. Approximately 109 pfu of fusion phages, W1 (E), W10 (‚) or1 (h), were reacted with various concentrations of lactose, and bound

actose was estimated from the pfu of bound phage as described underaterials and Methods. The ratios of the concentrations of bound lac-

ose to free lactose are plotted against the concentration of boundactose. Plots of the calculated concentration of bound lactose versusactose concentration added appear as saturation plots (inset). Pointsepresent the average of three assays and error bars are the standardeviation from the mean.

295

he reaction. Assuming that the concentration of thestimated bound phage is equal to that of bound lac-ose, dissociation constants were obtained from thelope of the Scatchard plots of the ratio of free to boundactose and the concentration of bound lactose (Fig.B). Dissociation constants for W1, W10 and C1, 3.7 3024 M, 5.0 3 1024 M and 4.0 3 1024 M, respectively,ere not significantly different from each other, indi-

ating that the lactose-binding domains with or with-ut the glycine and proline-rich tandem repeats havehe same affinity for lactose.

ISCUSSION

In this report, we have successfully defined the min-mal Gal-3 domain, from Ile-115 to Ile-250, essentialor lactose binding, using a bacteriophage l surfaceisplay, lfoo. During the course of this work, the crys-al structure of the Gal-3 lactose-binding domain haseen determined after removing its amino-terminalequence by collagenase digestion [22]. The amino-erminal residue Ile-115 of the domain may play atructural role for the protein folding of lactose-bindingomain, since the residue is not involved in directnteraction with the lactose molecule in the crystaltructure of the complex. An indispensable role for thearboxyl-terminal Ile-250 is also indicated by the crys-allography analysis of the protein, in which the elec-ron density of the terminal isoleucine residue islearly observed. The average resolution of the map-ing in this study was about seven amino-acid residuesn length, depending on the quality of the library used.herefore, the domain defined above may not be themallest region essential for lactose binding. Further-ore, the mapping result does not rule out a possibility

hat smaller Gal-3 fragments may have ability to bindactose, although respective deletion of 15 or 10 aminocid residues in length from the amino- or carboxylerminus of the minimal domain abolished its lactoseinding or stable expression in bacteria, respectively,s described earlier. Such smaller fragments may,owever, have less stable structures with lower affinityor lactose than the domain determined here. As de-cribed above, the domain identified in this work cor-esponds precisely to the folding domain of Gal-3 de-ermined by x-ray crystallography, thus providing strongupport for the validity of the phage display for mappingf domains involved in macromolecular interactions.Dissociation constants obtained using purified Gal-3olecules were on the order of 1025 M [ref. 15], an

rder of magnitude lower than the values obtained bysing the fusion l phage in this study. This is probablyecause in the present method the number of phageound to lactose may be underestimated. It was calcu-ated that the average number of Gal-3 fragments dis-layed is about five molecules per phage particle whenultivated with MC8 as described above. All the Gal-3

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ragments displayed on one phage particle might notind lactose and such phage might be counted as partf the unbound phage population. Therefore, it is rea-onable that the relative dissociation constants ob-ained by this method is higher than the values bio-hemically obtained by using the purified Gal-3 protein.

The method described here for the determination ofelative dissociation constants between fusion phagend ligand in solution does not require either purifiedr labeled proteins, and enables direct comparison ofhe binding affinity of individual phage clones. There-ore, this approach is much efficient than conventionaliochemical approach. However, dissociation constantsbtained in this way should be regarded as relativealues, since they are influenced by the presence ofultiple copies of fusion proteins on the phage. Rela-

ive dissociation constants between peptide fusionhage and specific antibodies to the peptides have beeneasured by using the filamentous phage fusion vector

29]. In this case, the values obtained are also relativeince the filamentous phage expresses multiple pep-ides per phage particle. However, proteins expressedn the surface of the phage may be mutagenized tosolate modified proteins that have various affinities toigand after affinity selection using ligand immobilizedo solid matrices such as microtiter wells and paramag-etic beads. Thus, the method described here may beseful for the studies of structure-function relationshipf various proteins.Lectin binding to polysaccharides is one of the weakest

mong various macromolecular interactions. Therefore,any macromolecular interactions including protein

inding to protein, DNA or RNA could potentially benalyzed by the approach we have used in this report.he strategy employed in this study will allow to effi-iently define the minimal folding domains of proteinsnvolved in various macromolecular interactions. Smallomains identified by this way should be advantageous toubsequent biochemical and structural analyses. For ex-mple, mutagenesis and x-ray crystallographic analysesf small domains may more efficiently be performed thansing whole protein molecules.

CKNOWLEDGMENTS

We thank H. Maruyama for technical assistance, and N. Kumar,. Niwa, and M. Kaneko for reading the manuscript. This work was

upported in part by NSF Grant MCB9424202 (I.N.M.) and NIHrant AI39620 (F.-T.L.).

EFERENCES

1. Scott, J. K., and Smith, G. P. (1990) Science 249, 386–390.2. Jespers, L. S., Messen, J. H., Keyser, A. D., Eeckhout, D.,

Brande, I. V. D., Gansemans, Y. G., Lauwereys, M. J., Vlasuk,G. P., and Stanssens, P. E. (1995) Bio/Technology 13, 378–382.

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Galfre, G., Pessi, A., Tramontano, A., and Sollazzo, M. (1995)Curr. Opin. Biotechnol. 6, 73–80.

4. Wilson, D. R., and Finlay, B. B. (1998) Can. J. Microbiol. 44,313–329.

5. Liang, O. D., Preissner, K. T., and Chhatwal, G. S. (1997) Bio-chem. Biophys. Res. Commun. 234, 445–449.

6. Jacobsson, K., Jonsson, H., Lindmark, H., Guss, B., Lindberg,M., and Frykberg, L. (1997) Microbiol. Res. 152, 121–128.

7. Kiewitz, A., and Wolfes, H. (1997) FEBS Lett. 415, 258–262.8. Malik, P., Terry, T. D., Gowda, L. R., Langara, A., Petukhov,

S. A., Symmons, M. F., Welsh, L. C., Marvin, D. A., and Perham,R. N. (1996) J. Mol. Biol. 260, 9–21.

9. Santini, C., Brennan, D., Mennuni, C., Hoess, R. H., Nicosia,A., Cortese, R., and Luzzago, A. (1998) J. Mol. Biol. 282, 125–135.

0. Maruyama, I. N., Maruyama, H. I., and Brenner, S. (1994) Proc.Natl. Acad. Sci. USA 91, 8273–8277.

1. Mikawa, Y. G., Maruyama, I. N., and Brenner, S. (1996) J. Mol.Biol. 262, 21–30.

2. Sternberg, N., and Hoess, R. H. (1995) Proc. Natl. Acad. Sci.USA 92, 1609–1613.

3. Dunn, I. S. (1995) J. Mol. Biol. 248, 497–506.4. Perillo, N. L., Marcus, M. E., and Baum, L. G. (1998) J. Mol.

Med. 76, 402–412.5. Hsu, D. K., Zuberi, R. I., and Liu, F.-T. (1992) J. Biol. Chem. 267,

14167–14174.6. Frigeri, L. G., Zuberi, R. I., and Liu, F.-T. (1993) Biochemistry

32, 7644–7649.7. Ackerman, S. J., Corrette, S. E., Rosenberg, H. F., Bennett, J. C.,

Mastrianni, D. M., Nicholson-Weller, A., Weller, P. F., Chin,D. T., and Tenen, D. G. (1993) J. Immunol. 150, 456–468.

8. Kuwabara, I., and Liu, F.-T. (1996) J. Immunol. 156, 3939–3944.9. Bresalier, R. S., Byrd, J. C., Wang, L., and Raz, A. (1996) Cancer

Res. 56, 4354- 4357.0. Dagher, S. F., Wang, J. L., and Patterson, R. J. (1995) Proc. Natl.

Acad. Sci. USA 92, 1213–1217.1. Vyakarnam, A., Dagher, S. F., Wang, J. L., and Patterson, R. J.

(1997) Mol. Cell. Biol. 17, 4730–4737.2. Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H.,

Barondes, S. H., and Rini, J. M. (1998) J. Biol. Chem. 273,13047–13052.

3. Kuwabara, I., Maruyama, H., Mikawa, Y. G., Zuberi, R. I.,Liu, F.-T., and Maruyama, I. N. (1997) Nature Biotechnol. 15,74–78.

4. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R.,Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239,487–491.

5. Liu, F.-T., Hsu, D. K., Zuberi, R. I., Hill, P. N., Shenhav, A.,Kuwabara, I., and Chen, S.-S. (1996) Biochemistry 35, 6073–6079.

6. Yang, R.-Y., Hill, P. N., Hsu, D. K., and Liu, F.-T. (1998) Bio-chemistry 37, 4086–4092.

7. Yanisch-Perron, C., Viera, J., and Messing, J. (1985) Gene 33,103–119.

8. Kuklinski, S., and Probstmeier, R. (1998) J. Neurochem. 70,814–823.

9. Dyson, M. R., Germaschewski, V., and Murray, K. (1995) NucleicAcids Res. 23, 1531–1535.