Ž .Journal of Immunological Methods 231 1999 119–135www.elsevier.nlrlocaterjim

Ribosome display: an in vitro method for selection and evolutionof antibodies from libraries

Christiane Schaffitzel, Jozef Hanes, Lutz Jermutus, Andreas Pluckthun )¨Biochemisches Institut, UniÕersitat Zurich, Wintherthurerstr. 190, Zurich CH-8057, Switzerland¨ ¨ ¨

Abstract

Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size,however, is almost always limited by the initial transformation steps following its assembly and ligation, as other allscreening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening andselection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems.We review here the principle and applications of ribosome display for generating high-affinity antibodies from complexlibraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA–ribosome–protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, verystringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of theinitial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By thismethod, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosomedisplay are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of theprotein species investigated and the objectives of the selection or evolution experiment. q 1999 Elsevier Science B.V. Allrights reserved.

Keywords: Ribosome display; scFv fragments of antibodies; In vitro selection; Directed evolution; Cell-free translation

AbbreÕiations: CDR, complementarity-determining region;DEPC, diethylpyrocarbonate; DTT, dithiothreitol; EDTA, eth-ylenediamine-tetraacetic acid; PCR, polymerase chain reaction;PDI, protein disulfide isomerase; RIA, radioimmunoassay; RT,reverse transcription; scFv, single-chain Fv antibody fragment;SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel elec-trophoresis; SELEX, systematic evolution of ligands by exponen-tial enrichment; V, immunoglobulin variable region; V , immuno-H

globulin heavy-chain variable domain; V , immunoglobulinL

light-chain variable domain; VRC, vanadyl–ribonucleoside com-plexes

) Corresponding author. Tel.: q41-1-635-5570; fax: q41-1-635-5712; e-mail: plueckthun@biocfebs.unizh.ch

1. Introduction

Kohler and Milstein established, more than 20¨years ago, a powerful method to produce monoclonalantibodies from immunized mice by fusing murineB-cells to myeloma cells. The advent of this hy-

Ž .bridoma technology Kohler and Milstein, 1975¨made possible the generation of a large number ofantibodies that are currently in widespread use inbiomedical research, diagnostics and more recently

Žalso in therapeutic applications Holliger and.Hoogenboom, 1998 . Hybridoma technology, how-

ever, is limited by several shortcomings. For in-stance, the target of choice to be used as the antigen

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00149-0

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135120

needs to be immunogenic, but must not be lethalwhen injected into animals. Furthermore, the anti-bodies produced are, by necessity, animal antibodies,which may cause immunogenic reactions when ad-ministered to humans. A way out of this particularproblem has been to resort to special mouse strainswhich are constructed to encode several human V

Ž .regions Lonberg et al., 1994; Jakobovits, 1995 .Nevertheless, while the specificities and affinities ofthe antibodies can be checked and antibodies can bechosen accordingly, they cannot be improved inhybridoma technology. The intrinsic affinity of anti-bodies obtained in vivo usually does not exceed 1010

y1 Ž .M Foote and Eisen, 1995 , and it is usually atleast one order of magnitude lower, as the generationof higher affinity molecules is not advantageousduring the immune response; and thus, there is noselection pressure for further affinity maturation.Thus, in order to obtain antibodies with improvedcharacteristics, it would be necessary to performchallenging cycles of structure-based engineering ap-proaches. However, the predictive accuracy of eventhe most sophisticated approaches is currently insuf-

Žficient for this task Yelton et al., 1995; Dougan et.al., 1998 . Therefore, evolutionary methods are, at

present, the only practical way to improve a givenmonoclonal antibody from a ‘‘normal’’ or a trans-genic mouse. Here, we describe a methodology to dothis.

The introduction of phage display technologyŽ .Smith, 1985; Scott and Smith, 1990 to the field ofantibodies, in conjunction with methods of generat-ing antibody libraries, allowed one to bypass some of

Žthese limitations McCafferty et al., 1990; Winter et.al., 1994 . In particular, the need for animal immu-

nization, as well as the lengthy process of repeatedimmunization, hybridoma generation and the screen-ing for specific antibodies was replaced by libraryconstruction and affinity selection. In phage display,the phage particles, produced by individual bacteria,contain the DNA sequence coding for the proteinthat is displayed on the phage surface, thus providing

Ž .a link between the genotype DNA sequence andŽ .the phenotype protein , which is a prerequisite for

any selection process. For instance, a single-chain FvŽ .scFv fragment of an antibody consisting of thecovalently linked variable domains of both heavy

Žand light chains Bird et al., 1988; Huston et al.,

.1988; Glockshuber et al., 1990 can be displayed onfilamentous phage as a fusion to a minor coat protein

Ž .from filamentous phage McCafferty et al., 1990and can be selected by binding to immobilized anti-gen. Alternatively, the display of Fab fragments ofantibodies on the surface of filamentous phages can

Žbe used Hoogenboom et al., 1991; Kang et al.,.1991 . In recent years, a wide array of antibodies has

been selected by phage display, utilizing large, naiveŽ .i.e., not stemming from preimmunized organismsrecombinant libraries of human antibody genesŽWinter et al., 1994; Vaughan et al., 1996; Aujame et

.al., 1997; Hoogenboom et al., 1998 or totally syn-Ž .thetic genes Knappik et al., manuscript submitted ,

thereby providing a convenient direct access to hu-man antibodies.

While phage display evidently represents a con-siderable progress over hybridoma technology, defi-ciencies still do exist. The necessary transformationstep limits the library size to approximately 1010

Ž .Dower and Cwirla, 1992 , and even this size re-quires a substantial investment of labor. Also, selec-tion in the context of the host environment cannot beavoided, possibly causing loss of potential candidatesdue to their growth disadvantage or even toxicity forEscherichia coli. Furthermore, difficulties in eluting

Žphages carrying antibodies with very high pico-. Žmolar affinity can be encountered Schier and Marks,.1996 . If a diversification step needs to be included

in the selection strategy in order to evolve the anti-body, either particular mutator strains can be usedŽ .Low et al., 1996 , that can also create unwantedmutations in the utilized plasmid and in the hostgenome, or it is necessary to repeatedly switch be-tween the selection procedure carried out in vivoŽ .phage, bacteria and the mutagenesis step for diver-

Ž w xsification polymerase chain reaction PCR or.recombination techniques in vitro. This is a particu-

larly laborious procedure, as after each diversifica-tion step, the newly created library has to be reli-gated and retransformed. Consequently, evolution ofantibodies over several cycles of diversification and

Žselection has only rarely been reported Yang et al.,.1995; Schier and Marks, 1996; Moore et al., 1997 ,

and it is not clear how this could ever become aroutine procedure.

Our laboratory developed recently a novel strat-egy for selection and evolution of antibodies entirely

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 121

Žin vitro — ribosome display Hanes and Pluckthun,¨.1997 . It is based on earlier studies with peptide

Ž .display Mattheakis et al., 1994 , but required con-siderable improvements in the display and selectionof folded proteins. In the improved ribosome display,many of the limitations of phage display are cir-cumvented by utilizing a cell-free transcription,translation and selection system. Using this newtechnology, it has been possible to select and evolvehigh-affinity antibodies with dissociation constants

y11 Ž .as low as 10 M Hanes et al., 1998 in a shorttime, starting from very large libraries which do nothave to be transformed into cells. In the followingsections, we describe the development and use ofribosome display, its present applications and futurepotential.

2. In vitro selection techniques

Ž .Tuerk and Gold 1990 introduced a technology,termed systematic evolution of ligands by exponen-

Ž .tial enrichment SELEX , by which it is possible toexponentially enrich and to evolve ligands consistingof RNA in multiple rounds in vitro from a randomoligonucleotide pool. Currently, this method is widelyemployed to screen for nucleic acid ligandsŽ .aptamers binding to numerous targets, and also toisolate novel, nucleic-acid-based synthetic catalystsfor a variety of potential applications in chemistry,

Ždiagnostics and, perhaps even therapy Gold et al.,.1995; Osborne et al., 1997 . In SELEX, multiple

rounds of in vitro transcription of random nucleicacid pools, affinity selection and reverse transcrip-

Ž .tion RT PCR are performed, thus giving rise toexponential enrichment of high-affinity specimens.The specific binders can subsequently be cloned andsequenced, and then characterized. The principle un-derlying SELEX is schematically depicted in Fig.1A. In SELEX, genotype and phenotype is repre-sented by the same RNA molecule, which exerts itsfunction through its three-dimensional structure,which in turn is determined by its nucleotide se-quence.

Nevertheless, RNA molecules have some severedisadvantages as ligands, and they have been essen-

tially replaced by peptides and proteins in evolution.For instance, RNA is extremely labile to ubiquitousRNases, and to actually use any aptamer in a realapplication requires first the stabilization of the RNA

Žutilizing stable nucleotide analogues Eaton et al.,. Ž1997; Ruckman et al., 1998 . The initial and re-

.maining appeal of SELEX was the very rapid gener-ation of high-affinity binders from very large initiallibraries. Since in ribosome display this problem hasnow been solved for peptides and proteins as well,the use of RNA aptamers appears less attractive incomparison. Already in their original publicationŽ .Tuerk and Gold, 1990 , the authors speculated thattheir methodology could also be adapted for proteinselection as well: Particular mRNAs had been iso-lated from a pool of variants by immunoprecipitationof the nascent polypeptides present in the mRNA–

Žribosome–polypeptide complexes Korman et al.,.1982; Kraus and Rosenberg, 1982 . In fact, a patent

Žapplication was filed soon afterwards Kawasaki,.1991 , proposing to utilize a similar approach to

enrich peptides from libraries. Mattheakis et al.Ž .1994 described the first experimental demonstra-tion of this technology, termed polysome display bythe authors. A synthetic DNA library encoding forpeptides was used in an E. coli coupled transcrip-tionrtranslation system. After in vitro transcriptionrtranslation, the mRNA–ribosome–peptide com-plexes were isolated by centrifugation with a sucrosecushion. The ribosomal complexes were subse-quently incubated together with the immobilized tar-get for affinity selection. After several washing steps,the mRNA of the bound mRNA–ribosome–peptidecomplexes was eluted with EDTA, reverse-tran-scribed and PCR-amplified. The PCR products couldbe used for subsequent selection cycles or for analy-sis of the pool for specific binders. A peptide withnanomolar affinity to an immobilized monoclonalantibody was selected by this technology from alibrary of 1012 molecules; however, the true diver-sity of the library was not analyzed. Subsequently,binders for a particular prostate cancer marker were

Ž .isolated using this technique Gersuk et al., 1997 .Several experiments showed co-translational fold-

ing of proteins to occur in in vitro translation sys-Ž .tems Fedorov and Baldwin, 1995, 1997 . Further-

more, the enzymatic activity of ribosome-boundŽnascent polypeptide chains Kudlicki et al., 1995;

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135122

A reverse transcription DNA

B

andPCR

/ ~~~ elution of bound "" RNA aptamers

immobilized target

DNA in vitro transcription ...........__.. mRNA

reverse transcnpt1on by PCR . . 1 mutations

and PCR errors ! in ~"" Uanolation

c

5'...........__..3' mRNA

i""alk>n of mRNA 1 5'~

•

DNA

reverse transcription 1 andPCR

5'

elution of the mANA protein

complex t

3' dissociation of the mANA­ribosome­

protein complexes

3• native protein,

- ./ tethered to

j ~ the

ribosome

selection with immobilized ligand (antigen)

3'

lll

in vitro transcription ...........__.. mRNA

r. vitro ligation

.._. DNA· puromycin linker

5'~

! in ~"" uano/ation

selection on 5' immobilized target r-.._.......__ __ , _ • A-site of

the ribosome

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 123

.Makeyev et al., 1996 could be detected, demonstrat-ing that release from the ribosome is not a pre-requisite for acquiring the native conformation of anascent polypeptide chain, provided folding of theprotein is not prevented by the ribosome tunnel.Thus, it appeared to be possible to obtain mRNA,ribosome and correctly folded functional protein in alinked assembly that could be used for screening andselection. The concept developed by Mattheakis et

Ž .al. 1994 was used for the development of ribosomedisplay of whole functional proteins in our labora-

Ž .tory Hanes and Pluckthun, 1997 .¨In ribosome display, a DNA library coding for

particular proteins, for instance scFv fragments ofantibodies, is transcribed in vitro. The mRNA ispurified and used for in vitro translation. As themRNA lacks a stop codon, the ribosome stalls at theend of the mRNA, giving rise to a ternary complexof mRNA, ribosome and functional protein. After invitro translation, the ribosomal complexes, which arestabilized by high concentrations of magnesium ionsand low temperature, are directly used for selectioneither on a ligand immobilized on a surface or insolution, with the bound ribosomal complexes subse-quently being captured, e.g., with magnetic beads.The mRNA incorporated in the bound ribosomalcomplexes is eluted by addition of EDTA, purified,reverse-transcribed and amplified by PCR. Duringthe PCR step, the T7 promoter and the Shine–Dalgarno sequence are reintroduced by appropriateprimers. Therefore, the PCR product can be directlyused for further selection cycles. Ribosome display isschematically depicted in Fig. 1B. The experimentalprocedures of this technique for the selection of scFvfragments of antibodies are commented on in detailbelow.

A 109-fold enrichment of scFv fragments of anti-bodies was achieved by five cycles of ribosomedisplay, utilizing an E. coli S30 extract for in vitrotranslation. Subsequently, it was demonstrated that itis possible to select and evolve scFv fragments ofantibodies from immune libraries by ribosome dis-play even with K values in the low picomolard

Ž .range Hanes et al., 1998 , and eukaryotic cell-freetranslation systems such as rabbit reticulocyte lysate

Žcan also be utilized He and Taussig, 1997; Hanes et.al., 1999 . Finally, the selection of high-affinity anti-

bodies from a library of synthetic antibody genes byusing ribosome display has now also been demon-

Žstrated Hanes, Schaffitzel, Jermutus and Pluckthun,¨.manuscript in preparation .

A somewhat different approach to couple pheno-type and genotype was designed by Roberts and

Ž .Szostak 1997 and independently by Nemoto et al.Ž .1997 , who linked a peptide covalently to its encod-ing mRNA. In this technology, called RNA–peptide

Ž .fusion Roberts and Szostak, 1997 or in vitro virusŽ . XNemoto et al., 1997 , mRNA is ligated at its 3 -terminus to a puromycin-tagged DNA linker in every

Ž .cycle Fig. 1C . The ribosome stalls upon reachingthe RNA–DNA junction, the puromycin enters theribosomal A-site, and the nascent peptide is therebycoupled to puromycin by peptidyl-transferase. Theresulting covalently linked complex of polypeptide,puromycin and mRNA–DNA hybrid can be dissoci-ated from the ribosome and used for selection exper-iments. This approach has so far only been demon-strated for peptides in a model enrichment. Thecovalent bond appeared attractive, when this methodwas first designed, as the link between peptide andmRNA seemed more stable than in ribosome display.However, we now know that the ternary ribosomal

Ž .Fig. 1. In vitro selection techniques. A SELEX. A DNA oligonucleotide pool is transcribed in vitro. The resulting RNA is directly used inŽ .affinity selection against an immobilized target. RNA molecules that bind termed ‘‘aptamers’’ are subsequently eluted. By RT-PCR, an

Ž .oligonucleotide pool enriched for binders can be regenerated and used for a new round of SELEX. B Ribosome display. A library ofproteins, e.g., scFv fragments of antibodies, is transcribed and translated in vitro. The resulting mRNA lacks a stop codon, giving rise tolinked mRNA–ribosome–protein complexes. These are used for selection on the immobilized target. The mRNA incorporated in boundcomplexes is eluted and purified. RT-PCR, which can introduce mutations, yields a DNA pool enriched for binders that can be used for the

Ž .next iteration. C Protein–RNA fusion. Covalent RNA–protein complexes can be generated by ligation of a DNA–puromycin linker to thein vitro transcribed mRNA. The ribosome stalls at the RNA–DNA junction. Puromycin then binds to the ribosomal A site. The nascentpolypeptide is thereby transferred to puromycin. The resulting covalently linked complex can be used for selection experiments.

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135124

complexes can be kept intact for at least 10 daysunder appropriate conditions, allowing very stringentoff-rate selections.

3. Ribosome display of scFv fragments of antibod-ies

3.1. The ribosome display construct

The scFv construct used for ribosome display isdepicted in Fig. 2. The construct does not contain astop codon, and thus a quite stable mRNA–ribo-some–polypeptide ternary complex can be formedduring in vitro translation, provided the complexesare stabilized by high Mg2q concentration and lowtemperature after translation. The 5X-untranslated re-gion of the ribosome display construct contains theT7 promoter for efficient transcription by T7 RNApolymerase. The 5X-untranslated region of the RNAis derived from gene 10 of phage T7 and containsthe Shine–Dalgarno sequence for efficient initiationof in vitro translation. This 5X-untranslated region iscapable of forming a stable stem-loop structure onthe mRNA level. The 5X-stem-loops were shown toprotect mRNA against degradation by RNaseEŽ .Bouvet and Belasco, 1992 . A second stem-loop

structure, present at the 3X-end of the mRNA, is ofparticular importance to protect mRNA from degra-dation by 3X-5X-exonucleases in the E. coli S30 ex-tract. The sequence for the 3X-stem-loop was derivedfrom the terminator of the E. coli lipoprotein or,alternatively, from the early terminator of T3 phage.The incorporation of both 5X- and 3X-terminal stem-loop structures in the construct led to an efficiency

Žgain of ribosome display by a factor of 15 Table 2;.Hanes and Pluckthun, 1997 . A variety of other¨

stem-loop sequences was also tested, but significantdifferences were not observed, leading to the conclu-sion that it is the existence rather than the sequenceof these secondary structures that is decisive.

The protein coding sequence consists of a proteinŽ .library and a spacer tether , which is necessary for

sufficient spatial separation of the nascent polypep-tide chain from the ribosome and thus enables thepolypeptide to fold correctly while being linked tothe mRNA–ribosome complex. It was demonstratedby various studies that the ribosome covers between20 and 30 C-terminal amino acids of a given

Žpolypeptide chain during protein synthesis Malkinand Rich, 1967; Jackson and Hunter, 1970; Smith et

.al., 1978 . Also, sufficient space and flexibility areprovided by this tether for the protein to recognizeand bind to the given target. Spacers derived from

Ž . Ž .Fig. 2. The scFv construct used for ribosome display. The promoter T7 is followed by a Shine–Dalgarno sequence SD and the scFvŽ .construct containing an N-terminal FLAG tag. Variable domains, V and V , are joined by a glycinerserine-rich linker. A spacer tether isH L

cloned in frame behind the sequence of the scFv fragment of an antibody without a stop codon. Sequences encoding for RNA stem-loopX X Ž .structures are present at the 5 - and 3 -ends. Characteristic restriction sites are indicated. In ribosome display, after the RT step primer T3te ,

Ž . Žtwo subsequent PCR steps are used to reintroduce the Shine–Dalgarno sequence PCR1; primers SDA, T3te and the T7 promoter PCR2;.primers T7B, T3te to regenerate a complete scFv construct.

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 125

gene III of filamentous phage M13 encoding 88–116amino acids were used for the display of scFv frag-

Ž .ments of antibodies Hanes and Pluckthun, 1997 ,¨and it was shown that the efficiency of ribosome

Ž .display increased with spacer length Table 2 . Forribosome display with oligopeptide libraries, a spacer

Žlength of 72 amino acids was used Mattheakis et al.,.1994 . For ribosome display with a rabbit reticu-

Ž .locyte lysate He and Taussig, 1997 and with an E.Ž .coli lysate Hanes et al., 1999 , the constant k-do-

Žmain of the light chain of an antibody He and.Taussig, 1997; Hanes et al., 1999 was also tested as

a spacer, with similar results. In ribosome displayutilizing E. coli cell extract for in vitro translation,spacers derived from the proline-rich sequence of

ŽTonB or the helical segment of TolA Pellitier et al.,.unpublished results , two proteins spanning the

periplasm of E. coli, were also used. However, thedifferences when using either one of these spacers,

Žcompared to the original gene III spacer Hanes et.al., 1999 , will require a more detailed investigation.

Furthermore, rare codons can be incorporated inŽ .the spacer region Mattheakis et al., 1996 , with the

intention of causing ribosome stalling and thus slow-ing down protein synthesis, perhaps additionally sta-bilizing the ribosomal complexes. However, a posi-tive effect of these rare codons has not yet been

Ž .experimentally demonstrated Table 2 .Any library can be assembled into the described

ribosome display format by ligation into the vector,Ž .pAK200 Krebber et al., 1997 , in frame with the

gene III spacer sequence in vitro, without transform-ing E. coli. The ligation mixture is then used di-rectly for PCR amplification. All necessary features,such as the Shine–Dalgarno sequence, the T7 pro-

moter sequence and the stem-loops are introduced inŽtwo subsequent PCR reactions for primers, see Table

.1 . As the PCR amplification terminates the genebefore the stop codon of gene III protein, the con-struct has no stop codon.

3.2. In Õitro transcription and translation

The initial DNA library is amplified by PCR andsubsequently transcribed by T7 RNA polymeraseand translated in vitro. In vitro transcription andtranslation can be carried out either in coupled or inseparate reactions. Proteins containing disulfidebridges, such as immunoglobulins, in general, foldcorrectly only under oxidizing conditions, such thatthe crucial intradomain disulfide bridges can be

Žproperly formed Goto and Hamaguchi, 1979;.Glockshuber et al., 1992 , and only a subset of

antibodies can fold in the absence of disulfide bondsŽ .Proba et al., 1998; Worn and Pluckthun, 1998a,b .¨ ¨On the other hand, T7 RNA polymerase requires

Žb-mercaptoethanol to maintain its stability Gurevich.et al., 1991 . Therefore, transcription and translation

should be carried out separately for scFv fragmentsŽ .of antibodies Hanes and Pluckthun, 1997 and other¨

proteins with disulfide bridges. However, a coupledrabbit reticulocyte system containing 2 mM DTT hasbeen used for display of scFv fragments of antibod-

Ž .ies He and Taussig, 1997 , but this approach wouldappear to be limited to the abovementioned subset ofantibodies. If proteins are employed in the selectionexperiment that can fold correctly under reducingconditions, a coupled in vitro transcriptionrtransla-tion system may be preferred.

Table 1Oligonucleotides used in ribosome display

Primer Sequence RemarksX X XT3te 5 -GGCCCACCCGTGAAGGTGAGCCTCAGTAGCGACAG-3 introduces the 3 -stem-loop derived from the

translated early terminator of phage T3;anneals to the gene III spacer

XSDA 5 -AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTT introduces the ribosome binding site;XAAGAAGGAGATATATCCATGGACTACAAAGA-3 used for the first PCR amplification step;

underlined is the NcoI restriction site for cloningX X XT7B 5 -ATACGAAATTAATACGACTCACTATAGGGAGACCACAACGG-3 introduces the T7 promoter and the 5 -stem-loop;

used for the second PCR amplification stepX XAnti-ssrA 5 -TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA-3 inhibits the 10S-RNA peptide tagging system

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135126

Conditions for efficient in vitro transcription haveŽ .been described in detail Gurevich et al., 1991 .

Approximately 0.1 mg mRNA is obtained after 2–3h in a 200 ml transcription reaction, utilizing 1–2 mgDNA template. The transcribed mRNA can be puri-fied by LiCl precipitation and a subsequent ethanol

Ž .precipitation Hanes et al., 1998 and then resus-pended in DEPC-treated water. Alternatively, RNApurification kits can also be used.

For E. coli in vitro translation, the preparation ofŽS30 extracts from E. coli MRE600 cells Wade and

.Robinson, 1966 is carried out following a modifiedprotocol, based on the procedure described by PrattŽ .1984 . In particular, the reducing agents, DTT andb-mercaptoethanol, are omitted from all buffers fordisplay of proteins containing disulfide bridges. TheE. coli system used for ribosome display needs to be

Ž .optimized according to Pratt 1984 with respect tothe concentration of Mg2q and Kq ions, the amount

Žof S30 extract used and the translation time Hanes.et al., 1999 . During in vitro translation, the protein

synthesis follows a saturation curve reaching aŽplateau after approximately 30 min Ryabova et al.,

.1997 . At the same time, mRNA is continuouslydegraded. Thus, an optimal time exists, at which theconcentration of intact mRNA–ribosome–proteincomplexes that can be used for selection is at amaximum. This optimal time for E. coli ribosomedisplay in vitro translation is usually between 6 and10 min after translation starts, as schematically de-

Ž .picted in Fig. 3 Hanes and Pluckthun, 1997 . Al-¨though most proteins generally fold more efficiently

Ž .at lower temperatures in vitro Creighton, 1994 , wefound that, at least for scFv fragments of antibodies,more ribosomal complexes containing functionalprotein were obtained when the reaction was carriedout at 378C, which may be attributed to the chaper-one activity in the E. coli extract. Endogenous tran-scription by E. coli RNA polymerase can be inhib-

Žited by addition of rifampicin Mattheakis et al.,.1996 . It is noteworthy that supplementing protease

inhibitors to the reaction did not have any effect, asjudged by 35S-methionine protein sodium dodecyl

Žsulfate polyacrylamide gel electrophoresis SDS-. ŽPAGE and autoradiography Hanes, Jermutus,

.Schaffitzel and Pluckthun, unpublished results . Also,¨prolonged incubation of the scFv fragments of anti-bodies in S30 cell extracts after translation did not

Fig. 3. Diagram showing the time course of protein synthesis andmRNA degradation during in vitro translation utilizing E. coliS30 cell extract and the resulting existence of a time optimum forintact mRNA–ribosome–protein complexes that can be used forselection. However, once the complexes bind to their immobilizedtarget and are washed, the RNases are essentially removed and theribosomal complexes are very stable.

change the amount of synthesized protein signifi-Žcantly, when analyzed by SDS-PAGE Hanes and

.Pluckthun, 1997; Hanes et al., 1999 .¨

3.3. Ribonucleases

In contrast to a coupled transcriptionrtranslationsetup, where a constant level of mRNA is maintainedthrough continuous transcription, particular attentionneeds to be paid towards RNases if the two stepshave to take place separately. E. coli MRE600 is astrain that lacks RNaseI, one of the predominantribonucleases that is involved in ribosomal RNA

Ž .degradation Meador et al., 1990 . To date, five ofthe 20 E. coli ribonucleases have been shown to

Žcontribute to mRNA degradation Hajnsdorf et al.,.1996 , many if not all of which are very likely to be

present in the S30 cell extract. Thus, mRNA stabilityis regarded as the limiting factor for in vitro transla-

Ž .tion Carrier and Keasling, 1997 . mRNA half lifecan vary from as short as 30 s to up to 20 min in E.coli, depending on mRNA secondary structure and

Ž .RNase activity Ehretsmann et al., 1992 . The RNaseŽ .inhibitor, VRC vanadyl– ribonucleoside complexes ,

a transition state analog, was shown to increase the

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Table 2Improvement of the efficiency of ribosome display

aFunction Effect in ribosome display

ConstructX X3 - and 5 -stem-loops stabilization against RNases 15-fold improvement

Stalling sequences in the spacer slows down the translation; not determinedŽ .Mattheakis et al., 1996 mRNA is protected by the ribosomesX2 -Modified pyrimidines incorporated mRNA stabilization nonein the mRNA

Phosphothioates mRNA stabilization noneVariation of the spacer length tethers the polypeptide to the ribosome; inceasing yield with increasing spacer lengthand sequence allows folding to a functional protein between 88 and 116 amino acids

TranslationAnti-ssrA oligonucleotide inhibition of the 10S-RNA peptide tagging system fourfold improvementProtease inhibitor cocktail protease inhibition None

Ž .without EDTA Boehringer MannheimPDI folding catalyst threefold improvementVRC RNase inhibition see text

Ž .RNasin Mattheakis et al., 1996 RNase inhibition requires reducing conditionsŽ . qPotassium glutamate 200 mM required to maintain a steady state K pool; several-fold improvement

Ž .instead of potassium acetate 100 mM glutamate is important in response to osmotic stressŽ .Cycloheximide Gersuk et al., 1997 translation stop and stabilization of the none in eukaryotic cell extracts

Ž .mRNA–ribosome–peptide complexes Jermutus, unpublished resultsin eukaryotic cell extracts

Ž .Chloramphenicol Mattheakis et al., 1996 translation stop and stabilization of the nonemRNA–ribosome–peptide complexes

Purification of the mRNA–ribosome–peptide removing RNases and proteases nonecomplexes by a sucrose cushion

Ž .centrifugation Mattheakis et al., 1996

a Ž . Ž .Data from Hanes and Pluckthun 1997 , Hanes et al. 1998; 1999 ; unpublished results.¨

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Žefficiency of E. coli ribosome display Hanes and.Pluckthun, 1997 , but also to inhibit the translation¨

Ž .process Table 2 . More recently, it has been omittedfrom the protocol, as it was found that better resultscan be achieved if the reaction mixture is cooledimmediately after stopping translation, and all subse-quent steps are meticulously carried out on ice. Also,it has proved beneficial to have all substances, buffers

Ž .and plastic precooled Hanes et al., 1999 . Further-more, the stem-loop structures present at the 3X- and5X-ends of the mRNA have improved the efficiency

Ž .of ribosome display by a factor of 15 Table 2 . The5X- and 3X-stem-loops may protect the RNA fromdegradation by exonucleases RNaseII and PNPase

X Žwhich act from the 3 -end of a message Hajnsdorf et.al., 1996 and against endonuclease RNaseE that

X Žrecognizes the 5 -end Bouvet and Belasco, 1992;.McDowall et al., 1995 . Fig. 4 schematically shows

the generally assumed mode for mRNA degradationŽ .in E. coli Carrier and Keasling, 1997 .

3.4. Stabilizing mRNA by incorporating modifiednucleotides

A further approach to stabilize mRNA involvesincorporation of modified RNA nucleotides during in

Ž .vitro transcription Aurup et al., 1992 . Besides us-ing modified nucleotides, post-transcriptional modifi-cation by acetylation of the 2X-hydroxyl groupsŽ .Ovodov and Alakhov, 1990 present in mRNA can,

in principle, have stabilizing effects against RNases.However, if not all nucleotides carry the substitutionin their sugar moieties, but instead only one or twoof the nucleotides have been replaced by modifiedones, stabilization of 2X-modified mRNA is, by ne-cessity, sequence-dependent. In this case, the tem-plate activity of modified mRNA is strongly depen-dent on which nucleotides were modified. Completesubstitution of the nucleotides with nucleotide ana-logues can actually inhibit in vitro translation en-

X Žtirely, if either 2 -modified residues Dunlap et al.,. Ž1971 or phosphothioates are used Ueda et al.,.1991 . We observed diminished in vitro transcription

and translation yields in our ribosome display experi-ments with 2X-amino- or 2X-fluoro-substituted pyrimi-dine nucleotides, while a significant stabilization ofthe modified mRNA against nucleolytic degradation

Žwas not observed Table 2; Schaffitzel et al., unpub-.lished results .

3.5. Protein expression and folding in ribosome dis-play

Selection of mRNA–ribosome–protein complexesdepends on correctly folded functional protein, in thepresent case scFv fragments of antibodies. It is well-established that antibody domains contain conserveddisulfide bridges which are critical for their integrityŽGoto and Hamaguchi, 1979; Glockshuber et al.,

.1992 . In the endoplasmic reticulum of eukaryotes,

Ž . XFig. 4. Model for mRNA degradation in E. coli. Exonucleases RNaseII, PNPase are inhibited by 3 terminal stem-loop structures on theŽ .mRNA. mRNA degradation is normally initiated by endonucleases RNaseIII, RNaseE and others that cut mRNA and can thereby remove

structured regions on the mRNA such as stem-loops. Resulting mRNA fragments can then be degraded further by exonucleases.

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 129

disulfide bond formation and isomerization is cat-alyzed by the enzyme protein disulfide isomeraseŽ . Ž .PDI Gilbert, 1998 in the presence of glutathioneŽ .Hwang et al., 1992 . In a systematic study of theeffect of PDI and chaperones on in vitro translationŽ .Ryabova et al., 1997 , it was found that disulfidebond formation and rearrangement needs to takeplace cotranslationally. In fact, superior yields offunctional antibody were obtained with eukaryoticPDI and a glutathione-redox shuffle. In the opti-mized batch system, about 8 mg of scFv antibodyfragment was synthesized in 15 minrml reactionvolume, with at least 50% of the protein folded tofunctional scFv. Molecular chaperones present in thecell extract such as DnaK, DnaJ, GrpE, andGroELrGroES, while increasing the solubility ofscFv fragments of antibodies to nearly 100%, did notinfluence significantly the net amount of functional

Ž .protein Ryabova et al., 1997 . We supplement ourin vitro translation reaction with eukaryotic PDI andare able to improve ribosome display efficiency

Ž .threefold Table 2; Hanes and Pluckthun, 1997 .¨

3.6. 10S-RNA

In E. coli, peptides synthesized from mRNAswithout stop codons are modified by the carboxy-terminal addition of an 11-amino-acid peptide tagŽ .AANDENYALAA , which is encoded by the 10S-RNA, the product of the ssrA gene. The first alanineis not encoded by ssrA, but is transferred to thenascent polypeptide from the tRNA moiety of 10S-

ŽRNA, whose aminoacyl residue is alanine Keiler et.al., 1996 . Addition of the peptide tag leads to

degradation by specific carboxy-terminal proteasesin the cytoplasm and periplasm. This system isthought to protect E. coli from conceivably harmfulpolypeptides derived from truncated mRNAs. Theprocess of tagging an mRNA without a stop codonby 10S-RNA also occurs during in vitro translationŽ .Himeno et al., 1997 . For this reason, we decided toblock the 10S-RNA system by means of an antisenseoligonucleotide complementary to the tag encoding

Ž .sequence of 10S-RNA Table 1 . Indeed, we ob-served a shortening of the longest translation productupon addition of this anti-ssrA oligonucleotide, con-sistent with the C-termini now being untagged, and a

concomitant fourfold efficiency gain of ribosomeŽ .display Table 2; Hanes and Pluckthun, 1997 .¨

3.7. Affinity selection

Translation arrest in ribosome display occurs byfourfold dilution of the translation mixture in anice-cold buffer containing 50 mM Mg2q. This mag-nesium concentration is used also throughout theentire selection process to stabilize the mRNA–ribosome–protein complexes. Mattheakis et al.Ž .1996 additionally supplemented the latter withchloramphenicol during the whole affinity selection.Chloramphenicol is thought to inhibit the elongationstep of translation, and Mattheakis et al. also used itto stabilize the ribosomal complexes. However, wedid not observe any beneficial effect of chloramphe-

Ž .nicol addition Table 2; Hanes and Pluckthun, 1997 .¨The diluted translation mixture can be used for

selection experiments directly without further purifi-cation of the ribosomal complexes by a sucrosecushion centrifugation as described by Mattheakis et

Ž .al. 1994 . The ribosomal complexes are stable for atleast 10 days at 48C; however, their amount gradu-

Žally decreases over this time Jermutus et al., unpub-.lished results . Selection is carried out in the pres-

Ž .ence of sterilized skim milk 1%–2% and heparinŽ .0.2% , which successfully prevent non-specificbinding of ribosomal complexes to surfaces andtherefore decrease the background signal. Heparinadditionally inhibits nucleases.

Selections can be performed both with ligandsimmobilized on a surface or in solution, if a taggedligand is used, which can then be captured, e.g., withmagnetic beads. Immobilization of protein antigenson plastic surfaces, however, may lead to partialunfolding of the protein due to hydrophobic interac-tions with the plastic. In extreme cases, this can leadto the selection of antibodies against protein epitopes

Žthat are not accessible in the native form Schwab.and Bosshard, 1992 . This can be circumvented by

selecting in solution. Here, a tag needs to be fused tothe antigen that allows subsequent capture of themRNA–ribosome–scFv complexes bound to theantigen. We used biotin-labeled antigen for selectionin solution which allows capture of the antigen-boundribosomal complexes on streptavidin-coated mag-

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135130

netic beads. It has been demonstrated previouslywith phage display that optimal selection of higher-affinity scFv fragments of antibodies can be achievedwhen selection is performed in solution against bio-

Ž .tinylated antigen Schier et al., 1996a,b . If biotin isused as a tag, the milk added to the selection reactionneeds to be depleted of biotin beforehand. We typi-cally alternate between streptavidin-coated magneticparticles and avidin–agarose, to avoid selectingstreptavidin or avidin binders.

The high stability of the ribosomal complexes at48C allows for intensive washing of the bound ribo-somal complexes with ice-cold Mg2q-containingbuffer. The desired mRNA species are then elutedwith EDTA-containing buffer. EDTA dissociates theribosomal complexes by chelating Mg2q. Alterna-tively, the bound ribosomal complexes can also beeluted by adding soluble, untagged antigen, whichoffers the advantage that non-specifically bound ri-bosomal complexes do not co-elute. However, high-affinity binders may be eluted less efficiently thanlower affinity ones. Cleavable linkers between anti-gen and biotin can also be used to elute ligand-boundmRNA–ribosome–scFv complexes. Nevertheless,the direct extraction of mRNA, which avoids all ofthese steps, is one of the advantages over phagedisplay in isolating very high-affinity binders.

The eluted mRNA is then purified with commer-cial mRNA purification kits, including DNaseI treat-ment to remove any DNA template bound non-specifically, which may still be present after in vitrotranscription, translation, selection and elution. Thepurified mRNA is utilized for RT-PCR and can alsobe used for Northern analysis to quantify the effi-ciency of the system. For RT-PCR, a primer is used

X Žthat anneals to the 3 -terminus of the mRNA Table.1 . The Shine–Dalgarno and T7 promoter sequences

at the 5X-end are reintroduced by subsequent PCRsteps with appropriate primers. Thus, only intactmRNA is reverse-transcribed and PCR-amplified.The PCR-amplified DNA can now be directly tran-scribed in vitro. The mRNA can either be used forthe next selection cycle, or be translated in vitro inthe presence of radioactive amino acids and tested by

Ž .radioimmunoassay RIA for specific binders presentin the pool. To obtain individual sequences, theDNA pool is ligated in a plasmid, transformed intoE. coli and the plasmids of single clones are isolated.

The individual clones can be analyzed by furtherRIA experiments and sequencing of single clones.Antigen-specific scFv fragments are generally en-riched by a factor of 100–1000 per cycle of ribo-

Žsome display Hanes and Pluckthun, 1997; Hanes et¨.al., 1999 . After five rounds, enrichments of up to

9 Ž10 have been achieved Hanes and Pluckthun,¨.1997 .

If large amounts of specific scFv fragments arerequired for further experiments, overexpression in

Žthe E. coli periplasm Skerra and Pluckthun, 1988;¨. ŽPluckthun et al., 1996 or in inclusion bodies Hus-¨

.ton et al., 1995 can be performed. Good yields offunctional scFv fragments, selected in vitro by ribo-some display, were observed on expression in E.

Ž .coli Hanes et al., 1998 , indicative of a selection forproteins that are expressed at a reasonable levelduring ribosome display using the S30 cell extracts.This also indicates that the fundamental rules forefficient protein folding must be the same in the celland in vitro.

4. In vitro evolution by ribosome display

If screening and selection of an unaltered libraryis required, proofreading polymerases can be usedfor the PCR amplification step in ribosome display.On the other hand, if a non-proofreading polymeraselike Taq DNA polymerase is used, which introduceson average one mutation per 20,000 nucleotidesŽ .Cline et al., 1996 , each selection round leads to adiversification of the DNA sequence pool. Thus,through combined mutation and selection, a processresembling affinity maturation of scFv fragments ofantibodies can occur. This was demonstrated whenselecting scFv fragments of antibodies against a mu-tant of the yeast transcription factor GCN4 from animmune mouse library. A monomeric scFv with a kd

of 4=10y11M was selected that apparently was notpresent in the initial immune library and had under-gone a 65-fold affinity maturation in the process of

Ž .ribosome display Hanes et al., 1998 . This suggeststhe enormous potential of ribosome display not onlyas a screening technique but also as an efficientmethod for real protein evolution.

Diversification during PCR can be further en-hanced by mutagenesis methods such as oligonucleo-

Ž .tide-directed mutagenesis Hermes et al., 1990 , er-

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 131

ror prone PCR in the presence of non-physiological2q Ž .metal ions such as Mn Lin-Goerke et al., 1997

Ž .or dNTP analogues Zaccolo et al., 1996 . OtherPCR methods that can be used for diversificationinvolve homologous recombination in vitro, such as

Ž . ŽDNA shuffling Stemmer, 1994 and StEP staggered.extension process; Zhao et al., 1998 . As both muta-

genesis and selection take place entirely in vitro,comparatively minor efforts are required to carry outmany evolution cycles. After a first selection of theinitial library, only the scFv encoding region isamplified and diversified in vitro. This second-gener-ation library is inserted into the ribosome displayformat by assembly PCR and subsequently tran-scribed into mRNA. The evolution of the sequencepool can be monitored by RIA. Since the ternaryribosomal complexes are stable for several days andselection pressure can be applied both during andafter translation, numerous applications for directed

Žprotein evolution are conceivable Jermutus et al.,.manuscript in preparation .

Recombination-based methods have the advantagethat deleterious or non-essential mutations can besuppressed by recombination with the original se-quence, while beneficial mutations persist and accu-

Ž .mulate Moore et al., 1997 . These methods canfurther be combined with random mutagenesis. Afurther approach to create mutant scFv repertoires isto replace the V or V genes of a set of antibodiesH L

Ž .with a V gene repertoire chain shuffling ; here, aŽsingle fixed cross-over site is utilized Schier et al.,

.1996a in contrast to DNA shuffling. These methodsare fully compatible with ribosome display and theprocedures can easily be incorporated into the proto-col.

5. Discussion and outlook

A cognate antibody is generally enriched 100-foldto 1000-fold during one cycle of ribosome displayŽ .Hanes and Pluckthun, 1997; Hanes et al., 1999 .¨The mRNA yield after in vitro translation, affinityselection and elution amounts to a total of 0.2%; i.e.,

Ž 13 .for every 10 mg mRNA about 1.5=10 moleculesinitially employed in a 100 ml in vitro translation

Ž 10 .reaction, 20 ng of mRNA about 3=10 moleculesencoding a specific antibody is recovered from ribo-

somal complexes. Therefore, in a 1-ml in vitro trans-lation reaction, a library of 3=1011 independentmembers is screened. Note, however, that the actualsequence space explored is much larger, because foreach selected variant, many thousand single, doubleand triple mutants are explored, due to the error rateof the polymerase employed. For phage display tosample the same sequence space, the single-pot li-brary would have to encode all these variants at onceand then be of a size of 1016, which is not feasible.

At mRNA-to-ribosome ratios close to those pre-Žsent in our ribosome display experiments approxi-

.mately 1:1 , predominantly monoribosomal com-Ž .plexes are obtained Hanes, unpublished results , as

evidenced by sucrose gradient centrifugation. Forthis reason, we prefer the term ‘‘ribosome display’’over ‘‘polysome display’’. A significant part of themRNA in the ribosomal complexes can be degradedby RNases. Also, some RNA molecules can be partof complexes with misfolded and thus non-functionalpolypeptides. For these reasons, in vitro translationconstitutes a critical step in ribosome display. Anunresolved issue is the presence of rather significantamounts of translation products shorter than ex-

Ž .pected Hanes and Pluckthun, 1997 , which are not a¨consequence of proteolytic degradation but possiblyof ribosome stalling prior to the end of the mRNA.After sucrose gradient centrifugation, the same pat-tern of radioactive-labeled shorter peptides was found

Žto be associated with ribosomes Hanes, unpublished.results . Still, some premature dissociation of either

the synthesized polypeptide chain from the riboso-mal complex or of the ribosome from the mRNAduring in vitro translation cannot be excluded. Inprevious studies, it was found that up to two thirds ofnascent polypeptide chains may dissociate during

Ž .translation Fedorov and Baldwin, 1998 . Dissoci-ated polypeptide chains could act as competitors tothe mRNA–ribosome–antibody complexes used forselection, if low amounts of antigens are used.Therefore, off-rate selections may be more advanta-geous than equilibrium competition for small amountsof antigen.

Recently, it was reported that condensation of E.coli cell extracts combined with dialysis during

Ž .translation continuous-flow system can increaseŽ .protein yields dramatically Kigawa et al., 1999 .

Yields of up to 6 mgrml protein were reported by

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135132

using this system. As was detailed above, significantimprovements of ribosome display efficiency can beachieved by utilizing more functional ribosomes andby variation and amelioration of in vitro translationconditions. Furthermore, by substituting potassiumacetate by potassium glutamate during in vitro trans-lation, we were able to considerably improve mRNA

Žyields during ribosome display Table 2; Hanes et.al., 1999 . Thus, it may well be that there are further

useful additives missing in the cell extract. By thesame token, other components may be present thatare inhibitory for the in vitro translation reaction.

By the absence of transformation steps in ribo-Ž 11.some display, very large libraries G10 have

become amenable for in vitro selection. Furthermore,ribosome display is not only a powerful screeningmethod, but simultaneously allows for sequence evo-lution by mutagenesis during the iterative selectionprocess. The combination of large library sizes andmutagenesis provides a possibility to cover a verylarge sequence space during evolution. Thus, syn-thetic combinatorial antibody libraries, which encode

Žthe entire structural human repertoire Knappik et al.,.manuscript submitted , can now be efficiently

screened by ribosome display, and a variety of high-affinity binders, which even have not been part of

Žthe initial library, have been selected for Hanes,Schaffitzel, Jermutus and Pluckthun, manuscript in¨

.preparation .Ribosome display can be utilized to obtain ultra-

Ž .high affinity picomolar and better antibodies sinceit is the mRNA–ribosome–antibody complex that isdissociated during the elution step rather than theantibody–antigen complex, as for instance in phagedisplay. Therefore, an elution is not absolutely nec-essary in ribosome display as the antibody can be‘‘left behind’’ — only the mRNA is needed. Byadjusting the selection conditions, it is possible togenerate not only high-affinity antibodies but also toimprove specificity for the recognized epitope, thestability of the antibody and other molecular proper-ties. The selection conditions can be further variedby adding competitor antigens and increasing selec-

Ž .tion time off-rate selection during the experimentŽ .Jermutus et al., manuscript in preparation .

In vitro translation conditions can also be variedin ribosome display. For example, the redox state ofthe system can be chosen by altered DTT. Chaper-

ones and enzymes that support folding such as PPI-Ž . Žase protein-prolyl cis–trans isomerase or PDI pro-

.tein disulfide isomerase can be added, and reactionconditions similar to those present in the periplasmor, alternatively, the cytoplasm of E. coli can becreated according to the needs of the selected proteinspecies. If disulfide-free functional antibodiesŽMarasco, 1995; Proba et al., 1998; Cattaneo and

.Biocca, 1999 for use as ‘‘intrabodies’’ need to begenerated, appropriate mutagenesis steps can becombined with ribosome display selection in an en-vironment similar to the E. coli cytoplasm. Theseintrabodies than can be expressed intracellularly tobind to their target and exert a variety of functions.

For selection experiments with certain proteins,requiring for instance the presence of eukaryoticchaperones, it may prove to be of value to testeukaryotic cell extracts such as rabbit reticulocytelysate or wheat germ extract for in vitro translation.We and others were able to demonstrate that forribosome display, both eukaryotic and prokaryotic

Žextracts can be used, each with its own merits He.and Taussig, 1997; Hanes et al., 1999 . So far, for

the antibody fragments tested, the E. coli extractŽproved to result in better enrichments Hanes et al.,

.1999 .

6. Conclusion

Ribosome display is a powerful method forscreening very large antibody libraries. Each step ofribosome display is carried out in vitro, thus circum-venting limitations associated with in vivo systems.Libraries can be further diversified during PCR stepsin ribosome display using low-fidelity polymerases.Thus, high-affinity antibodies initially not present inlibraries can be generated and selected against alarge array of antigens. Diversification can addition-ally be increased with mutagenesis and in vitrorecombination techniques that are well compatiblewith ribosome display, avoiding cumbersome alter-nations between in vitro and in vivo steps. Weanticipate that ribosome display will be of particularimportance in the future for directed evolution ofproteins through many generations, yielding versatilemolecules for a large variety of applications.

( )C. Schaffitzel et al.rJournal of Immunological Methods 231 1999 119–135 133

Acknowledgements

C.S. and L.J. are supported by predoctoral Kekulefellowships from the Fonds der Chemischen Indus-

Ž .trie FCI, Germany . The authors thank Imre Bergerfor critical reading of the manuscript.

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