Proc. Nati. Acad. Sci. USAVol. 91, pp. 9022-9026, September 1994Biochemistry

An in vitro polysome display system for identifying ligands fromvery large peptide libraries

(translation/recombinant diversity/amnity purification/protein synthesis)

LARRY C. MATTHEAKIS*, RAMESH R. BHATTt, AND WILLIAM J. DOWERAffymax Research Institute, 4001 Miranda Avenue, Palo Alto, CA 94304

Communicated by Masayasu Nomura, June 2, 1994 (received for review March 21, 1994)

ABSTRACT We have used an in vitro protein synthesissystem to construct a very large library of peptides displayedon polysomes. A pool ofDNA sequences encoding 1012 randomdecapeptides was incubated in an Escherichia coil S30 coupledtranscription/translation system. Polysomes were isolated andscreened by afflnity selection of the nascent peptides on animmobilized monoclonal antibody specific for the peptidedynorphin B. The mRNA from the enriched pool of polysomeswas recovered, copied into cDNA, and amplified by the poly-merase chain reaction (PCR) to produce template for the nextround of in vitro synthesis and selection. A portion of theamplified template from each round was cloned into a filamen-tous phagemid vector to determine the specificity of peptidebinding by phage ELISA and to sequence the DNA. After fourrounds of affinity selection, the majority of clones encodedpeptides that bound specifically to the antibody and containeda consensus sequence that is similar to the known epitope forthe antibody. Synthetic peptides corresponding to several ofthese sequences have binding affinities ranging from 7 to 140nM. The in vitro system described here has the potential toscreen peptide libraries that are three to six orders of magni-tude larger than current biological peptide display systems.

Peptide libraries derived from the cloning and expression ofrandom-sequence oligonucleotides provide a rich source ofligands. These libraries, which often contain >108 recombi-nants, are characterized by the physical linkage of eachpeptide to its encoding DNA. This feature permits the affinitypurification of peptides and associated DNA on an immobi-lized receptor and the DNA is sequenced to identify thepeptide. This general approach provides a powerful tool fordiscovering ligands.A widely used version of this strategy is the display of

peptides on the outer surface of filamentous phage particles.The peptide sequences are encoded by random oligonucle-otides inserted into the 5' region of the genes encoding thecapsid proteins pIII or pVIII (1-3). The peptides are ex-pressed on the phage fused to the N terminus of the coatproteins. Several rounds of screening and amplification resultin the enrichment of phage expressing peptides that bind tothe receptor. Another system employs peptides fused to theC terminus of the lac repressor Lacd (4). The repressorprotein physically links the peptides to the plasmid encodingthem by binding to the lac operator sequences on the plasmid.The peptide-LacI-plasmid complexes are screened in a man-ner analogous to the phage system.These library approaches to ligand discovery often rely on

the generation of huge numbers of peptides with the expec-tation that those ofthe appropriate structure will be rare. Thesize of a cell-based library is limited by the number ofrecombinants recovered from the transformation step. To

create a peptide display system that avoids this limitation andis capable of screening much larger libraries, we developedthe in vitro polysome system described here.

Studies have shown that mRNAs encoding abundant an-tigens can be enriched by immunoprecipitating polysomes(5-7). Tuerk and Gold (8) have suggested that the RNAlibraries they developed as a source of ligands could beredesigned and translated into libraries of nascent peptidesdisplayed on polysomes. Kawasaki (9) proposed a similarapproach in a patent application.Here we describe an in vitro system for displaying and

screening peptide libraries on polysomes (Fig. 1). We choseas our model receptor a monoclonal antibody (mAb; D32.39)that binds dynorphin B, a 13-residue opioid peptide (10).Previous studies using the Lacd fusion system have shownthat a 6-amino acid fragment of dynorphin B, Arg-Gln-Phe-Lys-Val-Val (RQFKVV), defines the linear epitope for mAbD32.39 (4). An Escherichia coli S30 system was incubatedwith DNA encoding 1012 decapeptide sequences, and poly-somes were isolated and screened for binding to D32.39.After four rounds of affinity selection, the majority ofmRNAsequences present in the pool encoded peptides that boundspecifically to the antibody and contained sequences similarto the epitope sequence. Several peptides from this groupwere chemically synthesized and shown to bind D32.39 withhigh affinity.

MATERIALS AND METHODSIn Vitro Synthesis and Isolation of Polysomes. The E. coli

S30 extract (Promega) was prepared from the B strain SL119as described (11). Synthesis reaction mixtures were in a finalvolume of 50 Al and included 20 Al of complete premix (orpremix lacking methionine for radiolabeling protein) (11), 15/il of extract, 1 /l4 of rifampicin (1 mg/ml), 100 units of T7RNA polymerase (Ambion, Austin, TX), 20 units of RNasin(Promega), and template DNA as indicated. Reactions wereincubated for 30 min at 370C and synthesis was stopped byplacing the mixture on ice and diluting it 1:4 with polysomebuffer [20 mM Hepes-NaOH, pH 7.5/10 mM MgCl2/chloramphenicol (1.5 tsg/ml)/acetylated bovine serum albu-min (100 pug/ml)/l mM dithiothreitol/RNasin (20 units/ml)/0.1% Triton X-100]. To radiolabel mRNA or protein, 5 ,uCi of[a-32P]UTP (Amersham, 3000 Ci/mmol; 1 Ci = 37 GBq) or[35S]methionine (Amersham, 617 Ci/mmol) was included inthe reaction and the incorporation of radioisotope was quan-titated by precipitating duplicate samples with 10o (wt/vol)trichloroacetic acid, measuring radioactivity in a liquid scin-tillation counter, and averaging the values. To isolate poly-somes, the diluted reaction mixtures were centrifuged at288,000 x g for 36 min at 40C, and the pellets were resuspended

Abbreviation: mAb, monoclonal antibody.*To whom reprint requests should be addressed.tPresent address: Department of Molecular Pharmacology, StanfordUniversity School of Medicine, 300 Pasteur Drive, Stanford, CA94305.

9022

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Proc. Natl. Acad. Sci. USA 91 (1994) 9023

Clone PT7 (NNK)n-and

Sequence Synthetic DNAlibrary

7

6 Amplification

_ CoupledTranscription! Translation

5- (NNK)-

3- n 5' (NNK)- 3 5

cDNA5 Synthesis

Elution ofL@ i | mRNA

4

2

5' (NNK)n An; ... 3

----(NNK)n- --- 3' lAffinitySelection 3

ImmobilizedReceptor

FIG. 1. An in vitro polysome system for screeningpeptide libraries. Steps: 1, a synthetic DNA librarycontaining a randomNNK codon region is incubated inan E. coli S30 coupled transcription/translation sys-tem; 2, protein synthesis is stopped with chloramphen-icol and polysomes are isolated by centrifugation; 3,polysomes are added to wells containing an immobi-lized receptor for affinity selection; 4, the bound poly-somes are dissociated with EDTA and the mRNA isrecovered; 5, the mRNA is copied into cDNA; 6, thecDNA is amplified by PCR using primers that restorethe T7 promoter (Pr7); 7, a portion ofthe enriched poolis cloned into a phagemid vector for ELISA andsequencing before repeating the cycle.

in polysome buffer and centrifuged a second time at 10,000 xg for 5 min to remove any insoluble material. To measure theincorporation of mRNA into polysomes, equal amounts of32P-labeledmRNA reaction products were diluted in polysomebuffer or elution buffer (polysome buffer plus 20 mM EDTA)and centrifuged as described above. The fraction of totalmRNA that was specifically released from polysomes byEDTA was determined by trichloroacetic acid precipitation.

Affinity Selection of Polysomes. Microtiter wells (Corning)were prepared by incubating each well with 5 ug of mAbD32.39 in PBS (10 mM sodium phosphate, pH 7.4/140 mMNaCl/2.7 mM KCl) for 1 hr at 370C, washing with PBS,blocking with PBS/1% nonfat milk for 1 hr at 370C, andwashing again with polysome buffer. Polysomes were added,as indicated, and incubated with the immobilized antibody for2 hr at 40C. Each well was washed five times with 100 ,ul ofpolysome buffer and the mRNA was eluted in 100 jLd ofelution buffer for 30 min at 40C.

Screening of a Polysome Library. Polysomes were isolatedfrom a 50-j4 reaction mixture programmed with 440 ng ofDNA library and equal portions were added to six microtiterwells containing the immobilized mAb D32.39. After affinityselection, the recovered mRNA samples were combined andtreated with 6 units of DNase I (Ambion) for 15 min at 370Cafter increasing the MgCl2 concentration to 40 mM. ThemRNA was phenol-extracted and ethanol-precipitated in thepresence ofglycogen, and the pellet was resuspended in 20 glof RNase-free water. A portion of the mRNA (8.5 Ild) washeated for 3 min at 800C and chilled on ice, and 50 pmol ofprimer ON1914 (5'>GATTGTGGAAGCTTGGCGCCTGCT-3') was added tossynthesize cDNA with the avian myeloblas-tosis virus reverse transcription system (Promega). ThecDNA was amplified by PCR in a reaction mixture consistingof 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100,2.5 mM MgCl2, all four dNTPs (each at 0.5 mM), 5 units ofTaq polymerase (Promega), and 0.5 juM primer ON1415containing the T7 promoter (5'-ACTTCGAAATTAATAC-GACTCACTATAGGGAGACCACAACGGTTTCCCTCT-3'), and 0.5 ,M primer ON1230 (5'-GGCGCCTGCTGCCT-GCGTGTCGCCTGTCGT-3'). Amplification consisted of 30cycles at 950C for 45 sec and 720C for 1 min. The amplifiedproduct was gel-purified and quantitated by measuring theA260.DNA Sequencing. Single-stranded phagemid DNA was

isolated by Prep-A-Gene (Bio-Rad), and the random regionwas sequenced using the Sequenase system (United StatesBiochemical).Phage ELISA. For ELISA, the microtiter wells were pre-

pared as described above except that 1 pg ofmAb D32.39 perwell was used and the blocking buffer consisted of PBS/1%bovine serum albumin. Duplicate portions of phage culturesupernatant (50 ILI) were added to wells and incubated for 2

hr at 40C. Wells were washed 10 times with 250 .d ofPBS, and100 1d of horseradish peroxidase conjugated to sheep anti-M13 IgG (1:2000 dilution, Pharmacia) was added and incu-bated for 1 hr at 40C. Wells were washed with PBS andbinding was detected by adding substrate [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium (0.2 mg/ml) 50 mM citric acid, pH 4/0.05% hydrogen peroxide] andmeasuring the A405. Phage clones were scored as positive ifthe average A405 value was at least 2-fold greater than thatobtained for binding to wells not coated with the mAb.

Determination of Peptide Binding Affinities. Peptides weresynthesized using standard Fmoc/t-Bu protection and hy-droxybenzotriazole-based coupling, purified to >90% byHPLC, and confirmed by mass spectrometry. The competi-tion binding assay was performed as described (12) andincluded a low concentration (50 pM) of the tracer peptidecontaining the D32.39 epitope sequence.

RESULTSConstruction of a Synthetic Gene for Expression of Peptides

in Vitro. A synthetic gene for expressing N-terminal peptidesunder the control of the bacteriophage T7 promoter wasconstructed. Oligonucleotide cassettes were ligated to uniquerestriction sites of the T7 expression plasmid pT7-7 (13) (Fig.2). Cassettes encoding the D32.39 epitope sequence (MAL-QFKVVT, epitope sequence underlined) or a scramblednonbinding control sequence (MAVFKRTVQ) were ligatedin-frame to a repeating Gly-Ser coding region. The 31-residueGly-Ser sequence provides a flexible spacer for presentingthe nascent peptide to the receptor. When these plasmids arelinearized with HindIII, the predicted gene product is aprotein of 93 residues with either the epitope or controlsequences beginning at amino acid position 3. There are nostop codons in any of the three possible reading frames.In Vitro Expression of Nascent Peptides and Binding of

Polysomes to mAb D32.39. Several factors were consideredimportant for developing the in vitro polysome display sys-tem. A significant fraction of the total mRNA should beassociated with polysomes at the end ofthe incubation periodand the nascent peptides must be accessible for binding to thereceptor. The polysome complexes and encoding mRNAsmust remain stable during the isolation and affinity selectionprocedures, and the in vitro system should be efficient inprotein synthesis.We chose the E. coli S30 system for in vitro expression

because it translates mRNA with high efficiency, is wellcharacterized, and is a coupled system that supports bothtranscription and translation (11, 14, 15).We next determined the specificity of polysome binding to

mAb D32.39 by the nascent peptide. Plasmids encoding theepitope (pLM138) or control sequences (pLM142) were lin-earized with HindIII and incubated in separate S30 reaction

Biochemistry: Mattheakis et al.

9024 Biochemistry: Mattheakis et al.

T7 promoterACTTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC 50

rbs NdeITAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCTCGTC 100

1 M A R QEcoRI

AGTTCAAAGTTGTTACCGAATTCTCCGGCAGCGGTTCCGGCAGCGGTTCC 1505 F K V V T E F S G S G S G S G S

GGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGG 20021 G S G S G S G S G S G S G S G S G

BamHICAGCGGTGGATCCTCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTC 250

38 S G G S S A A V P A A V P A A V PSalI

CGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTGTCG 30055 A A V P A A V P A A V P A A V S

ACAGAAGAAGGAGAAGGAGAAGGAGAAGGAGAAGGACGACAGGCGACACG 35071 T E E G E G E G E G E G R Q A T R

HindIII ClaICAGGCAGCAGGCGCCAAGCTTATCATCGAT

88 R Q Q A P S

A 1 0,000 -

8,000 -

EC.,,,: 6,000-z

ECD 4.000

2,000

371

FIG. 2. Nucleotide sequence and predicted amino acid sequenceofthe D32.39 epitope fusion protein gene. Nucleotides are numberedon the right; amino acids are numbered on the left. The gene wasconstructed by annealing synthetic oligonucleotides to their com-plementary strands to generate double-stranded cassettes flanked bythe indicated restriction sites. Individual cassettes were cleaved bythe appropriate restriction enzymes and subcloned sequentially topT7-7 starting with the Sal 1-HindIII cassette and followed by theBamHI-Sal I and EcoRI-BamHI cassettes. The Nde I-EcoRI cas-settes were subcloned last and contained either the D32.39 epitopesequence (underlined) or the control sequence, 5'-CATATIGCT-GTTTTCAAACGTACCGTTCAGjAATC-3' (Nde I and EcoRIsites are underlined). The overlined sequences indicate the T7promoter, gene 10 ribosome binding site (rbs), and indicated restric-tion sites.

mixtures containing [a-32P]UTP to label the newly synthe-sized mRNA. Translation elongation was stopped by addingchloramphenicol and the mixtures were centrifuged to pelletpolysomes and free ribosomal subunits. Radiolabeled poly-somes containing the epitope or control coding sequenceswere added to separate microtiter wells containing the im-mobilized mAb D32.39. After affinity selection, EDTA wasadded to dissociate the antibody-bound complexes and thelabeled mRNA was recovered. The amount ofmRNA recov-ered from the wells was linear with increasing polysome input(Fig. 3A). Polysomes containing mRNA encoding the epitopebound at -10-fold higher levels than control polysomes, andbinding was blocked by the prior addition of free dynorphinB peptide to the wells (Fig. 3B). This indicates that bindingof polysomes to mAb D32.39 is peptide-specific.

Construction and Characterization of a DNA Library Con-taining a Random Population of Decapeptide Sequences. Apopulation of DNA molecules encoding random decapep-tides was constructed (Fig. 4). A degenerate oligonucleotidewas synthesized that contained 10 codons of the form NNK,where N represents equimolar A, C, G, or T and K isequimolar G or T. There are 32 possible codons resultingfrom the NNK motif: 12 amino acids encoded by uniquecodons, 5 amino acids each encoded by 2 codons, 3 aminoacids each encoded by 3 codons, and one stop codon (amber).A unique segment of this oligonucleotide mixture was an-nealed to an oligonucleotide containing the T7 promoter,extended with DNA polymerase, and ligated to a DNAfragment containing the Gly-Ser coding region by using BstXIsites. The ligated product, forming the DNA library, wasgel-purified and quantitated.To assess the randomness of the library, a portion of it was

cloned into the filamentous phagemid vector pAFF6, and therandom regions of 20 clones were sequenced (Fig. 5). Thedistribution of amino acids appeared to be random and noneof the sequences were similar to the known dynorphin Bepitope sequence (data not shown).The mass ofDNA library added to the in vitro system was

optimized by measuring the incorporation of [35S]methio-

B 1,200-

1,000 -

E& 800-

zg 600-E-)

400-w

200 -

0-

200 400 600Polysome input, cpm x 10-3

.IT I/VT VFKRTVQ

800

FIG. 3. Specific binding of polysomes to mAb D32.39. Radiola-beled polysomes were isolated from reaction mixtures programmedwith 1.5 ug of HindIII-linearized plasmid pLM138 or pLM142 andbound to microtiter wells containing the immobilized mAb. Therecovered mRNA was quantitated by trichloroacetic acid precipita-tion. (A) Binding of polysomes containing the D32.39 epitope (U) orcontrol (E) sequences. (B) Competition binding assay. Microtiterwells were preincubated with polysome buffer in the absence (solidbars) or presence (open bars) of 10 AM dynorphin B peptide for 1 hrat 4°C before adding polysomes (131,000 cpm) containing the D32.39epitope (RQFKVVT) or control (VFKRTVQ) sequences. The errorbars indicate the standard error of the duplicate samples. In aseparate experiment, the total amounts of peptide fusion proteinsynthesized from pLM138 and pLM142 were found to be approxi-mately equal (data not shown).

nine into protein. Incorporation of label in a 50-,ul reactionmixture was directly proportional to the amount of addedDNA up to z400 ng (1012 molecules). The amount ofradiolabeled mRNA synthesized from 400 ng of DNAlibrary was -3 mol of mRNA per mol of DNA (data notshown).To measure the incorporation of library mRNA into poly-

somes, we determined the fraction of total mRNA that wasreleased from polysomes after treatment with EDTA. Equalamounts of radiolabeled mRNA (5287 cpm) from an in vitroreaction mixture were centrifuged in the absence or presenceofEDTA and the amounts that cosedimented with ribosomeswere determined to be 1637 cpm and 198 cpm, respectively.We estimate that 27% of the total mRNA pool is present inpolysomes [(1637 - 198)(100)/5287].

Selecting Peptide Ligands from a Polysome Library. Wescreened polysomes expressing a random population ofdecapeptides for binding to mAb D32.39. An in vitro systemwas programmed with 1012 molecules of DNA library and

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 9025

DNA Pool

ON154351 P7 , BstXl

TUIL (NNM)10 mON1747 BstXl BstXl

I Extend and Cut with BstXlcut with B13i0 and gel purify

NNK CCAGGGCG TTGG Gly-Ser CCACAATC(NNK)10- GGTC CCGCAACC GGTG

Ligate and gelpurify product

M A S X10 G T Q G V G...GGAGATATACATATGGCTAGC(NNK) GGTACCCAGGGCGTTGGA...

Nhel 10 KpnIpill processing site

S H S M A S G T G G G G STCTCACTCCATGGCTAGCTAATAGTGGCCAGGATAGGTACCGGCGGTGGCGGCAGTL-_ _ Nhel Msc/ KpnI

pAFF6

BstXIPT7 Gly-Ser CCACAATC~~(NNK)I0 GGTG

FIG. 4. Construction of a DNA library encoding a randompopulation of decapeptide sequences. The degenerate region wasconstructed by annealing 100 pmol of oligonucleotides ON1543[containing the T7 promoter (Pr7) and nt 1-90, Fig. 2] and ON1747[5'-AAATTTCCAACGCCCTGGGTACC(MNN)ioGCTAGCCAT-ATGTATATCTCCTTCTT-3', M = A or C] and extending in areaction mixture containing 104 units of Sequenase (United StatesBiochemical), all four dNTPs (each at 1 mM), and 10 mM dithio-threitol for 30 min at 37eC. The extended product was cleaved withBstXI, ethanol-precipitated, and resuspended in water. The BstXIfragment containing the Gly-Ser coding region shown on the rightwas prepared by digesting plasmid pLM145 with BstXI and gel-purifying the 277-bp fragment. Plasmid pLM145 was constructed byinserting BstXI site linkers between the HindIll and Cla I sites andNde I and EcoRI sites ofpLM142. Approximately 4 pg of the Gly-Serfragment was ligated to an equivalent amount of the degenerateregion in a reaction mixture containing 400 units ofT4 ligase, 50 mMTris HCl (pH 8), 10mM MgCl2, 10mM dithiothreitol, 1mM ATP, andbovine serum albumin (25 pg/ml) for 16 hr at 150C. The 411-bp ligatedproduct (267 kDa or 2.5 x 1012 molecules per pg) was gel-purified andquantitated.

incubated, and polysomes were isolated and added to micro-titer wells containing the immobilized mAb D32.39. Afteraffinity selection, the bound mRNA was recovered, copiedinto cDNA, and PCR-amplified by using primers that in-cluded the sequences for the promoter region of T7 RNApolymerase. Approximately 400 ng of the amplified DNAproduct was then added to the S30 system for a subsequentround of in vitro synthesis and affinity selection.

After each round of selection, a portion of the amplifiedDNA template was cloned into pAFF6 and the random regionwas sequenced and compared to the known 6-residue epitopeand related sequences that had been identified using the Lacdfusion system (Fig. 5) (4). The relative frequency of se-quences similar to the epitope sequence was 2/6, 6/13, 16/19,and 9/9 after rounds 2-5, respectively (Fig. 6). The mosthighly conserved residues are an invariant Arg at position 1and Phe at position 3. Most of the clones (52%) contain thepositively charged residues Lys, Arg, or His at position 4.The aliphatic residues Val, Ile, Leu, and Ala are the mostfrequent group of amino acids found at positions 5 (76%) and6 (71%), with Val the preferred residue. No strong bias wasevident for residues in position 2. These sequences aresimilar to those found using the Lacd system except atposition 3, where Phe occurred in only 35% of the LacI-positive sequences (4). One sequence (PIM&SEFKXYL, epi-tope-like sequence underlined) that contains a sequencediffering from the natural epitope sequence only at position2 occurred in five of the nine clones recovered from round 5.

FIG. 5. Cloning of the DNA pool into the phagemid vectorpAFF6 for sequencing and ELISA. Approximately 25 ng oftemplateDNA from the original library and after each round of templateamplification was cleaved with Nhe I/Kpn I and ligated to the samesites of pAFF6, resulting in translational fusions of library peptidesto the pII capsid protein of M13 (C. Wagstrom and S. Cwirla,personal communication). Individual clones were isolated aftertransforming E. coli ART 293 [HfrCprIA8914 zhc::TnlO(tets, kanr) thirecA::cat] and grown in LB medium (10 g of tryptone/5 g of yeastextract/5 g ofNaCl per liter) containing glucose (0.1%) and ampicillin(100 pg/ml). At a cell density of 2 x 108 cells per ml, 1 x 1010transforming units of VCSM13 helper phage and kanamycin (20pg/ml) were added, and the cultures were induced by addingarabinose (0.02%) and grown overnight to isolate recombinant phageas described (16).

The binding specificities of these peptides for mAb D32.39were determined by ELISA. The assembled phagemid par-ticles display the polysome-derived peptides as N-terminalfusions to the capsid protein pll. The N-terminal sequenceof the processed recombinant pIll is identical to the poly-some-derived sequence for the first 15 residues (Fig. 5). Wetested each unique peptide clone that contained a sequencesimilar to the epitope by phage ELISA. All ofthe clones werepositive in the ELISA test except for sequences HNEGIML-E&YY, GMYETRLFHYQ, and FSERRFSVCW. Thesethree sequences are clearly related to the ELISA-positivesequences, but they contain 29 nt in the random regioninstead of 30 nt resulting in a frameshift mutation of thedownstream pIl sequence (data not shown). Since the Gly-Ser coding region of the polysome expression gene containsno stop codons, the in vitro system can apparently enrich forsequences of various lengths despite changes in the readingframe.

Binding Affinities of Enriched Peptides for mAb D32.39.Peptides corresponding to six ofthe enriched sequences werechemically synthesized and purified, and their identity wasconfirmed by mass spectrometry. A competition bindingassay was used to estimate their affinity for the mAb D32.39under conditions in which the IC50 value should approximatethe Kd value. The binding affinities of the six peptides rangedfrom 7.2 to 140 nM (Fig. 6). One of the ELISA-negativesequences (FSERRESVYCW) was also synthesized and foundto bind with an affinity of 110 nM. The authentic dynorphinB peptide had an IC50 value of 0.29 nM in this assay.

DISCUSSIONWe have created a huge library of L-amino acid peptides byexpressing 1012 DNA sequences in an E. coli S30 coupled

Biochemistry: Mattheakis et al.

9026 Biochemistry: Mattheakis et al.

Dynorphin B Y G G F L RJR Q F K V VIT

Round Frequency2 1

1

3 13011

4 3b

12111

11111

la115 5bl

ic1

K S L W R P F A Q VW Q T R R F S V A S

L R E F R C V MH N E G I R M F R V V

Y L R P F R V T FN H W R P F K T V I

P I

G M YM T

D SS Y

H N E

DELDRSSpG

NYDSVTKYTTCRI

P IMK R FF S E

DE F

R S F K V VR I F K I IR Q F S I CR L F K C VR P Y R L VR L F H V GR P F M V TR Q F S V TR Q F S V VR L F A Q VR L F R I VR H F Q I VR M F R V V

R S F K V VR M F K L VR R F S V CR Q F S I CR M F A V A

LQTVQ

RVA

LVwTC

IC50 (nM) each molecule of DNA and 27% of the mRNA pool is

0.29 + 0.05 specifically associated with polysomes after the 30-min in-cubation period. We believe that our method can be modifiedto express larger libraries of 1014_1015 DNA molecules. TheS30 reaction volume could be increased 100-fold or thesystem could be supplemented with some S30 componentthat may be limiting the formation of polysomes.

Y M 100 i 39 The diversity of peptide sequences expressed in vitroshould exceed that ofcell-based systems. The transformation

v efficiency and capacity of E. coli limits the practical size ofclone libraries to 109-1010 recombinants (18). By avoiding

7.2 ± 0.75 bacterial transformation for peptide expression, the in vitroP T 19 ± 1.2 system has the potential to generate libraries containing twoD H 140 ± 41 to six orders ofmagnitude more peptides. In addition, certainC sP cellular processes such as secretion and proteolysis that may

limit the diversity of cell-based libraries are absent or dimin-L ished in the in vitro system, and there is no requirement forN biological activity of the peptide-protein fusions. It shouldK also be possible to create libraries containing unnatural amino

acids by supplementing the in vitro system with suppressortRNAs that have been chemically acylated as described (19).

7.2 ± 0.75 Finally, in vitro expression of libraries provides the op-8.0 ± 0.22 portunity to continuously introduce variation into the se-110 ± 40 quence pool by low-fidelity amplification between rounds of

D H 140 ± 41 selection. This technique has been used to create RNAenzymes that exhibit new or enhanced catalytic functions (20,21). Repeated mutagenesis coupled with affinity-selective

FIG. 6. Amino acid alignment of selected peptide sequences withdynorphin B. The 6-residue D32.39 epitope sequence ofdynorphin Band the peptide regions similar to it are shown within the box. A totalof 6, 13, 19, and 9 clones from rounds 2-5, respectively, weresequenced. The frequency indicates the number of times eachsequence occurred among the clones picked from each round, andthe superscript letters a, b, and c indicate identical sequences foundin different rounds. Binding affinities for D32.39 were determined bychemically synthesizing the indicated peptide sequences and mea-suring the IC50 value. The IC50 values represent the mean ± SEM ofthree experiments.

transcription/translation system. Polysome complexes con-sisting of nascent peptides linked to their encoding mRNAswere stabilized with chloramphenicol and screened for bindingto an immobilized antibody that recognizes dynorphin B andrelated peptides. The bound complexes were dissociated withEDTA, and the encodingmRNAs were copied into cDNA andPCR amplified to produce DNA templates. Several rounds ofin vitro synthesis and selection resulted in pools of enrichedsequences that were cloned into a phagemid vector to deter-mine the specificity ofpeptide binding by phage ELISA and tosequence the DNA. By this process, we recovered a family ofrelated peptides that bind specifically to mAb D32.39 and aresimilar in sequence to the known epitope.The binding affinities for six ofthe recovered peptides range

from 7 to 140 nM. In contrast, the phage and Lacd fusiondisplay systems can recover ligands with affinities as low as100 ,uM due to the multivalent display of the peptides (ref. 17;Ron Barrett, personal communication). Peptide valency onpolysomes can vary according to the number-ofribosomes thatinitiate at each mRNA and that have synthesized a nascentchain of sufficient length for binding. We have not yet explic-itly determined the number of peptides displayed on eachpolysome complex, but the selection of ligands in the lownanomolar range suggests that very few peptides are accessi-ble on each polysome. Our repeated recovery of the 7 nMpeptide (PIMRSFKVVL) in five of the nine clones pickedfrom the fifth round is consistent with this possibility.The peptide library reported here is derived from the in

vitro expression of 1012 DNA molecules. This quantity oflibrary DNA (400 ng) does not saturate the transcriptional ortranslational capacity of a 50-il S30 reaction mixture. Ap-proximately three molecules of mRNA are synthesized for

screening should provide the rapid evolution and identifica-tion of peptide ligands of high affinity. Libraries of peptidesexpressed in vitro and displayed for affinity selection onpolysomes offers significant potential advantages over thepowerful peptide display techniques now in use.

We thank Chris Wagstrom for constructing the pAFF6 vector,Sherril Johnson for technical assistance, truce Mortensen for pro-viding mAb D32.39, the DNA Core Group for oligonucleotidesynthesis and DNA sequencing, and Ron Barrett, Steve Cwirla,Peter Schatz, and Pim Stemmer for helpful discussions.

1. Cwirla, S. E., Peters, E. A., Barrett, R. W. & Dower, W. J. (1990)Proc. Natl. Acad. Sci. USA 87, 6378-6382.

2. Scott, J. K. & Smith, G. P. (1990) Science 249, 386-390.3. Devlin, J. J., Panganiban, L. C. & Devlin, P. E. (1990) Science 249,

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