Surface presentation of protein epitopes using bacteriophage expression systems George P. Smith University of Missouri, Columbia, Missouri, USA Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying those phage whose displayed foreign peptide binds to an antibody or another binding protein. Present libraries display only short random peptides, but work is presently underway to create libraries displaying antibodies with a great diversity of binding specificities. Current Opinion in Biotechnology 1991, 2:668-673

Introduction

ands for receptors, enzymes, viruses, etc. might lead to the development of novel drugs or vaccines.

In the past few years, several forms of a new kind of expression library, which can generically be called alfinityselectable libraries (ASLs), have been developed. Each clone in an ASL consists of a genetically coded ligand (either a (poly)peptide or nucleic acid) attached to a replicon [which could be a phage, a cell, or a nucleic acid amplifiable by polymerase chain reaction (PCR)] containing the information specifying the ligand. ASLs can be searched for ligands that bind any given ligate (antibody, receptor, transcription factor, or other specific binding protein) in a simple way: the ligate is used to atfinity-purify the binding ligands and the attached replicons are then either propagated en masse in preparation for another round of afflnity selection, or cloned and propagated separately for individual analysis. Because the search is conducted on all clones en masse, vast libraries--10 TM or more clones in some systems - can be effectively surveyed. This is an increase of at least three orders of magnitude over the number that can be screened with conventional expression vectors. This great increase in scale permits new kinds of libraries to be conceived. For the first time we can contemplate what might be called an 'all-purpose' library: one so vast and varied as to contain specific ligands for almost any ligate of a certain type. With such a system, no advance knowledge of a ligate's specificity would be required to use it to affinity-purify binding clones and thus identify ligands, possibly including ligands that could never have been predicted from knowledge of the ligate. Because of the central role of specific binding in so many aspects of biology, an 'all purpose' ligand library would be a powerful new tool with many uses in basic and applied research. It might provide an entirely new way of studying the specificity of binding. The identification of novel lig-

Several realizations of the ASL concept have been explored, differing in the nature of the ligand and the mode of propagation; five such examples are shown in Table 1. This review concentrates on systems in which the ligands are foreign peptides or proteins fused to coat proteins of the F-pilus-specific filamentous bacteriophage (the class that includes the familiar strains M13, fl, and fd). These expression vectors are termed fusion phage [1,2].

Filamentous fusion phage In a filamentous fusion phage library each clone is a fusion phage that displays on its surface either a foreign peptide or a protein domain fused to one of two phage coat proteins: either plII, five copies of which are incorporated into one tip of the long, thin virion; or pVIII, several thousand copies of which comprise the bulk of the tubular coat. The gene for the fusion protein residues inside the infectious particle as part of the single-stranded viral DNA (ssDNA). Affinity-purified phage retain their infectivity, and can thus be cloned and propagated simply by infecting fresh host cells. The amino acid sequence of the displayed peptide is readily ascertained by sequencing the relevant portion of the viral ssDNA from individual clones. Both pill and pVIII are synthesized with amino-terminal signal peptides, which are cleaved off concomitantly with translocation of the polypeptides through the inner membrane of the envelope. Eventually they are anchored to the inner membrane by a single membrane-spanning domain, their carboxyl termini in the cytosol and their

Abbreviations ASL--affinity-selectable library; CDR--complementarity determining region; ssDNA--single-strandedDNA; PCR--polymerase chain reaction. 668

~) Current Biology Ltd ISSN 0958-1669

Surface presentation of protein epitopes using bacteriophage expression systems Smith 669 Table1. Examplesof affinity-selectable constructs. Propagatable unit COS cell filled with shuttle plasmid [18] DNA or RNA [19] Escherichia coil with

plasmid [20,21] bacteriophage*

Filamentous phage

Mode of propagation

Ligand Mammalian cellsurface proteins

Transfectionof E. coli with plasmid

Part of DNA or RNA

PCR

Foreign polypeptides fused to LamB or PhoE

Bacterialgrowth

Foreign (poly)peptide fused to £V or D protein

Infection of

Foreign (poly)peptide fused to pill or pVlll

Infection of

E. coli

E. coli

"M Koob, J Badger and W Szybalski, personal communication. PCR, polymerase chain reaction.

amino termini, including the fused foreign (poly)peptide, in the periplasm. Subsequently, the proteins are transferred from the membrane into the nascent virion as it emerges through the envelope. This membrane-bound mode of capsid assembly neither kills the cell nor prevents cell division. There is a prima facie resemblance between translocation of the coat proteins into the periplasm and translocation of eukaryotic membrane-bound or secreted proteins into the lumen of the endoplasmic reticulum. In particu-

lar, eukaryotic proteins with disulfide bonds can at least sometimes fold correctly when directed to the periplasm of Escherichia coll. It is not entirely unexpected, therefore, that entire folded protein domains, such as growth hormone [3"] and the Fv and Fab domains of antibodies [4.,--6-.], can be displayed on the virion surface. Several new fusion phage libraries and constructs have been introduced just in the past year (Table 2). In the rest of this section I shall review what I see as the most important issues in design of fusion-phage systems. (For a general review of filamentous phage physiology see [7] and for a review of the use of these phage as vectors see

[8].) One-gene versus two-gene systems In a one-gene system, the fusion protein is the only pIII or pVIII protein available for incorporation into the virion. In a I two-gene system, the fusion protein is supplemented witli the corresponding wild-type protein, so that the particles bear a mixture of coat proteins, only some displaying the foreign domain. The advantage of the two-gene system is that the presence of wild-type protein permits fusions that partially impair coat-protein function and for that reason would not be tolerated in one-gene systems.

Phage versus phagemid Ordinarily, propagation of filamentous phage requires multiple rounds of infection. Thus, partial deficits in pin or pVIII function, incurred as a result of long foreign inserts, severely depress plaque formation and overall yield. These partial deficiencies can also indirectly affect yield as a result of cell killing: cells harboring the defective phage die after little or no phage production. For these rea-

Table 2. Filamentous fusion phage constructs. Name of vector or construct

Phage gene

fUSE5 [14o'] fAFF1 [15"] M13LP67 [13oo] fd-CAT1 [4.0]

I11 111 III III

phGH-M13glll [3"']

Ill

pSEX [5..]

III

pCBAK8 [6,o] fdH [12oo]

VIII VIII

pKfdH [12oo]

VIII

*Amino acid sequences given in the single letter code.

Insert

Type of system

Random hexapeptide library Random hexapeptide library Random 15-mer library Single-chain Fv domain fused to full-length pill Human growth hormone fused directly to carboxyl-terminal domain of pill Single-chain Fv domain fused to full-length pllN Fab domain fused to pVlll Peptides NANP* and YGFWGM* fused to pVlll Malaria peptides NANPNANPNANP* and NDDSYIPSAEKI* fused to pVlll

One-gene, fd-tet phage One-gene, fd-tet phage One-gene, phage One-gene, fd-tet phage Two gene, phagemid

Two-gene, phagemid Two-gene, phagemid One gene, phage Two-gene, plasmid

670

Expressionsystems sons, conventional phage vectors will probably be suitable only for rather short peptide inserts. Vector fd-tet, on the other hand, has been able to accommodate foreign inserts as long as 250 amino acids in one-gene constructs [4..]. The use of vector fd-tet has diminished some of the dependence on pill and pVIII function, both because it greatly reduces cell killing and because it has a tetracycline resistance gene that allows phage to be propagated, in the same was as plasmids, independently of phage assembly and infectivity [9]. A single infection event by an fd-tet phage is sufficient to give a detectable, propagatable tetracycline-resistant clone. Theoretically, at least, propagation and phage functions can be even further divorced by transplanting the hybrid coat-protein gene to a phagemid [10,11] - - a plasmid containing the intergenic region of filamentous phage in addition to a conventional replication origin and an antibiotic resistance gene. The intergenic region contains all the ci~acting elements that form the sites of initiation of replication and the morphogenetic signal, vc~ch are required for ssDNA synthesis and packaging into virions. When a cell harboring a phagemid is superinfected by a helper phage that supplies all the phage proteins, it secretes not only helper virions but also infectious phagemid virions. The latter are readily distinguished because they are able to confer antibiotic resistance on any new host cell they infect.

G e n e III fusions

There are five copies of pill at one tip of the virion. The amino-terminal portion is exposed as a knob flexibly attached to the bulk of the particle. The protein is centrally involved not only in virion assembly but also in infectivity. Most fusion phage place the foreign residues at or near the amino terminus of mature pill, just downstream of the signal peptide (Table 2). Long foreign inserts ( > 100 amino acids) can sometimes reduce infectivity [2] and, less frequently, reduce the yield of physical particles. Both effects could possibly stem from the degradation of the recombinant protein [2]. These deleterious effects of foreign inserts can be ameliorated by two-gene systems. The low number of foreign domains displayed by the phage, especially in two-gene systems, can be an advantage, because it reduces multivalent binding and thus potentially enhances the ability to select for the highest amm~ty [3"]. In the two-gene system constructed by Bass et al. [3"], a large foreign domain (growth hormone) replaces the amino-terminal knob of plII, which, although essential for morphogenesis, is not required for incorporation into the virion. Removal of the amino-terminal domain might facilitate proper folding of the foreign domain, and also ensures that phagemid-bearing cells can be readily infected by helper phage, because the amino-terminal domain is primarily responsible for the inability to superinfect infected cells.

Gene VIII fusions There is one pViII molecule for approximately every 2.4 nucleotides of DNA, which corresponds to 2700 copies in wild-type particles. The amino terminus of the protein is exposed to the medium, and fusions have been placed slightly downstream of the signal peptide. Only very short inserts, of up to six amino acids, are tolerated in one-gene constructs [12-.], but much larger inserts can be accommodated in two-gene systems. Greenwood et al. [12..], for example, displayed 500-800 copies of foreign peptide epitopes along the length of the particle; in another example, Kang etaL [6"] displayed between one and 24 functional copies of a 50 kD antibody Fab domain. These phage do not appear to have impaired infectivity, which would perhaps be expected as presumably the only role of pviII in infection is the negative one of releasing the ssDNA as it enters the cell. The high copy number of the foreign domains in gene VIII fusions might be a great advantage in some circumstances; for instance, Greenwood and coworkers [12"] showed that phage displaying hundreds of copies of a foreign peptide epitope served as a potent immunogen for eliciting antibody against the foreign epitope.

Affinity selection The most commonly used procedure is 'biopanning' [2], in which a biotinylated ligate is first reacted with the library in a small volume (e.g. 10 IA), and is then diluted and 'panned' for a few minutes on a streptavidin-coated polystyrene petri dish. The dish is washed to remove unbound phage, and the bound phage are then eluted, still in infectious form, in acid. A library can also be panned using a petri dish that has been directly coated with the ligate [6",13.']. Ligate can be coupled covalently at high density to small, impermeable polyacrylic oxirane beads, greatly increasing the ratio of surface area to working volume [3"]. Sepharose beads have also been used as solid support [4",5"], but it is not entirely clear how they work as it seems unlikely that phage can penetrate far into the interior of the beads where most of the ligate molecules presumably reside.

Peptide libraries: first try for 'all-purpose' libraries Three 'epitope libraries' have been constructed in which the foreign insert is a degenerate oligonucleotide-- that is, a mixture of millions or billions of sequences synthesized in a single run on the chemical synthesizer by incorporating mixtures of monomers at selected positions [13"-15"']. Each phage thus displays a short peptide encoded by one component sequence in the oligonucleotide mixture, the library as a whole representing millions of peptides. When antibodies that are known to be specific for short peptide epitopes are used to survey these libraries, the affinity-purified clones nearly always express peptides that are identical or closely related to

Surface presentationof protein epitopes using bacteriophage expressionsystemsSmith the immunizing peptide epitope (JK Scott and GP Smith, unpublished data) [14--,15..]. From this, I conclude that as long as the peptide library contains high-affinity ligands for an antibody, that antibody will select some highalTmityligands from the library. The majority of anti-protein antibodies are not directed at short linear peptide epitopes, however; they recognize 'assembled' epitopes composed of residues that are distant in the primary sequence but adjacent in the folded antigen protein. The use of antibodies against assembled epitopes enables us to test whether short, random peptide libraries qualify as the 'all-purpose' ligand libraries mentioned earlier. If so, these antibodies should find high-affinity 'mimotopes' (a short peptide that mimicks an assembled epitope [16]) in the library. This, however, is not always the case OK Scott and GP Smith, unpublished data). We find that only about half these antibodies can identify mimotopes, and that all mimotopes have a weaker binding afi~nityfor their antibodies than genuine linear epitopes. Antibodies (or other ligates) that are specific for non-proteinaceous ligands can also sometimes identify peptide mimics. For instance Devtin et aL [13 °°] have recently shown that streptavidin binds to clones displaying a short peptide sequence motif within a 15-residue random sequence; the peptides seem to be mimics of biotin, as the latter blocked binding of phage to streptavidin. Presumably the short peptides displayed on these epitope libraries are very flexible. In one sense this is an advantage, in that a single peptide sequence effectively represents a multitude of molecular shapes. But this multiplicity of shapes is bought at the price of weakening the aiTmity for any given ligate, which will only bind a very limited subset of the conformations available to an unconstrained peptide. Perhaps, then, the first-generation peptide libraries erred too far on the side of flexibility, and performance can be improved by constraining the conformation of the random peptides in some w a y - - for example, by introducing disulfide bonds or by displaying random peptide loops in the context of a rigid protein framework. Indeed, we already know of a highly successful all-purpose ligand library with the latter design - - antibodies. The binding moiety of antibodies is the Fv domain which consists of the heavy (H) and light (L) chain variable regions. Structurally it consists of a rigid scaffolding with six highly variable peptide loops, the complementarity determining regions (CDRs), lining one of its faces. It is this lining that is chiefly responsible for the binding atTmityof an antibody.

'Phage antibodies': monoclonals of the future? In the past year, three groups have constructed filamentous phage displaying functional antibody domains on the surface. Two of these 'phage antibodies' display a single-chain Fv domain that is a single polypeptide comprising the H- and L-chain variable regions linked by a short nondescript peptide [4..,5.°]. The other displays a

two-chain Fab domain [6 ,o] in which the H-chain moiety, which comprises the variable region and the first subdomain of the constant region, is fused to pVIII; the L-chain is synthesized separately and must associate with the phage-bome H-chain in the periplasm before or concomitantly with viral assembly. All three groups present evidence that the phage-bome antibody retains the antigen-binding specificity of the parent monoclonal antibody. This work on a few specific phage antibodies is the first step toward an extraordinary goal. In a few years, it is hoped, researchers will be able to purchase aliquots of phage-antibody libraries representing a vast diversity of binding specificities. The researcher will use any antigen of interest to alt~nity-select phage displaying an antibody specific for that antigen. By analogy with at~nity maturation in the immune system, al~nity and specificity might be improved by random mutagenesis followed by another round of affinity selection. These will be the monoclonals of the future, obtained by simple microbiological manipulations, without the need for animals or animal cells in culture. Affinity selection is at the core of this project, and two of the groups have reported mock selections, in which they recover phage-antibody from a mixture containing a large excess of wild-type phage by afflnity-purifyingwith immobilized antigen [4",5"]; the wild-type phage, which have no displayed antibody domain, stand in for the background of non-binding phage-antibodies that would be present in a library. In light of our own experiments with an anti-fluorescein phage-antibody (EB Watson and GP Smith, unpublished data), however, caution should be taken in interpreting such enrichment experiments. Although afi~nity purification with immobilized fluorescein enriched our phage-antibody by 5000-fold over wildtype phage, we observed 100-fold enrichments in three critical negative controls: substitution of rhodamine for fluorescein, introduction into the antibody domain of a single amino acid substitution that abolished fluorescein binding, and competition with a large excess of free fluorescein hapten. The residual enrichment observed in the negative controls suggests that a certain degree of non-specific 'stickiness' of at least some antibody-phage occurs (perhaps because of incorrect folding of some fraction of the foreign antibody domains) indicating that wild-type phage are not an adequate negative control for non-specific binding. Other phage antibodies of different specificity are a somewhat better negative control [6"'], although even here it should be borne in mind that different antibody domains might exhibit different degrees of non-specific stickiness. How is the diversity of binding specificities in these antibody libraries to be achieved? Two strategies have been proposed. The first I call the 'natural repertoire' strategy. Its aim is to clone the diverse variable region repertoire that actually exists in immunocompetent animals. This is a natural extension of the 'combinatorial' antibody libraries that have already been constructed in conventional phage £ expression vectors [17]. Phage anti-

671

672

Expression systems bodies displaying Fab domains [6"] would be a convenient system for this type of library, because the H- and L-chain portions are coded separately, as they are in the immune cells that will be their source. The altemative approach, which ! call the random CDR strategy, starts with a single well-behaved antibody domain (for example, one that exhibits a particularly low amount of nonspecific stickiness) and then substitutes randomized peptides in place of the antibody's CDRs. Single-chain Fv constmcts [4o.,5",] would seem to be most convenient for this type of library. The premise of the random CDR approach is that the power of the immune system does not lie in any particular repertoire of antibodies. Rather, it is the vast scale of that repertoire that is important, coupled with the ability of the immunizing antigen to select from that repertoire those species that bind it most efficiently. These two features (vast scale and powerful selection) are precisely the distinguishing virtues of the surface expression system I have reviewed.

5. •.

BREITLINGF, DUBEL S, SEEHAUST, KLEWINGHAUSI, ~ M: A Surface Expression Vector for Antibody Screening. Gene 1991, 104:147-153. The same single-chain antibody Fv that was used by McCafferty et aL [4 ° ' ] is fused to pIII in a two-gene, phagemid construct. As in the case of [4,*] the specific enrichment experiments suffer from use of wildtype phage as background control. This would be a good vector for 'random CDR' libraries. 6. °o

KANG AS, BARBAS CF, JANDA KD, BENKOVIC SJ, LERNER RA: Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces. Proc Natl Acad Sci USA 1991, 88:4363-4366. Two-gene, phagemid constructs displaying antibody Fab domains along the length of the virion. The H-chain moiety of the Fab is fused to gene VIII, while the L-chain is synthesized separately, and must associate with the phage-borne H-chain to reconstitute whole Fab. This would be a convenient vector system for 'natural repertoire' libraries. 7.

MODELP, RUSSEL M: Filamentous Bacteriophage. In The Bacteriophage, Vol 2 edited by Calendar R [book]. New York: Plenum Publishing Corporation 1988, pp 375-456.

8.

SMITH GP: Filamentous Phages as Cloning Vectors. In Vectors.. A Survey of Molecular Cloning Vectors a n d Their Uses edited by Rodriguez RL, Denhardt DT [book]. Boston: Butterworths 1987, pp 61--83.

9.

SMITH GP: Filamentous Phage Assembly: Morphogenetically Defective Mutants that do not Kill the Host. Virology 1988, 167:156-165.

10.

~ , ~ DA, KEMPERB: Chimeric Single-stranded DNA Phageplasmid Cloning Vectors. In Vectors: A Survey of Molecular Cloning Vectors a n d Their Uses edited by Rodriguez RL, Denhardt DT ]book]. Boston: Butterworths 1987, pp 85-102.

11.

CESARENIG: Phage-plasmid Hybrid Vectors. In Vectors: A Survey of Molecular Cloning Vectors a n d Their Uses edited by Rodriguez RL, Denhardt DT ]book]. Boston: Butterworths 1987, pp 103-111.

Acknowledgements Preparation of this review and unpublished work reported therein were supported by NIH grant GM41478 and the Molecular Biology Program of the University of Missouri. I thank A Kang, C Barbas, M Little, W Szybalski and R Perham for showing me manuscripts in advance of publication or telling me of unpublished work in progress.

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1.

SMITH GP: Filamentous Fusion Phage: Novel Expression Vectors that Display Cloned Antigens on the Surface of the Virion. Science 1985, 228:1315-1317.

2.

PARMLEYSF, SMrrH GP: Antibody-selectable Filamentous fd Phage Vectors: Affinity Purification of Target Genes. Gene 1988, 73:305-318.

3. •°

BASS S, GREENE R, WELLSJ& Hormone Phage: An Enrichment Method for Variant Proteins with Altered BInding Properties. Proteins Struct Funct Genet 1990, 8:309-314. A two-gene construct containing growth hormone (a 190-residue protein with two disulfides) fused to the carboxy-tel~inal domain of gene RI on a phagemid. This report shows by far the most convincing demonstration that small protein domains can fold properly on the surface of phage. MCCAFFERTYJ, GRIFFITHS AD, WINTER G, CHISWELLDJ: Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains. Nature 1990, 348:552-554. The first phage antibody to be reported. A single-chain Fv domain with specificity for hen-egg lysozyme is fused to plII in an fd-tet-based, onegene construct. On ELISA the phage-antibody binds hen but not turkey lysozyme, providing good evidence for at least some specific binding by phage-borne antibodies. Their specific enrichment experiments suffer from the use of wild-type phage as background control. This would be a good vector system for 'random CDR' libraries. 4. °°

12. °.

GREENWOODJ, WILLIS AE, PERHAM RN: Multiple Display of Foreign Peptides on a Filamentous Bacteriophage: Peptides from P l a s m o d i u m f a l c i p a r u m Circumsporozoite Protein as Antigens, J Mol Biol 1991, 220:821-827. Very short peptides are fused to pVII1 in one-gene constructs in wildtype fd phage, and are displayed In thousands of copies along the length of the virion. Longer peptides are fused to pVIII in two-gene, plasmid constructs; superinfection with helper phage leads to the formation of hybrid phage that display hundreds of copies of the peptide. These phage are potent immunogens for eliciting antibody against the displayed peptide, a feature that might be extremely useful in rapid vaccIne development programs. 13. °°

DEVLINJJ, PANGANIBANLC, DEVL1N PE: Random Peptide Libraries: a Source of Specific Protein Binding Molecules. Science 1990, 249:404-406, Reports a one-gene, random 15-met library in a an M13 vector. Streptavidin is used to atfinity-select clones, which turned out to share a sequence motif at various positions in the 15-residue variable segment. 14. SCOTTJK, SMrrH GP: SearchIng for Peptide Ligands with an o° Epitope Library. Science 1990, 249:386-390. Random hexapeptides are fused to plII in a one-gene, fd-tet-based, 2 x 108 clone 'epitope library'. Anti-peptide monoclonal antibodies select phage-bearing peptides that have close sequence sinlilarity to the immunizing epitope. 15. °®

CWIRIASE, PETERS EA, BARRETTRW, DOWER EJ: Peptides on Phage: a Vast Library of Peptides for Identifying Ligands. Proc Natl Acad Sci USA 1990, 87:6378-6382. Reports a one-gene, fd-tet-based random hexapeptide 'epitope library' very similar to that constructed by Scott and Smith [14°°]. Again, an antipeptide antibody selects phage-bearing peptides that are similar but not identical to the immunizing epitope. The peptides are synthesized chemically and shown to have dissociation constants rather higher than that of the immunizing epitope, indicating that afflnity-selection under their conditions may not necessarily identify the tightest ligands.

Surface presentation of protein epitopes using bacteriophage expression systems Smith 16.

GEYSENHM, RODDASM, MASONTJ: The Delineation of Peptides Able to Mimic Assembled Epitopes (with Discussion). In Synthetic Peptides as Antigens edited by Porter R, Whelan J [book]. New York: Wiley 1986, pp 130-149.

20.

CHARBIT A, MOLLAA, SAURINW, HOFNUNGM: Versatility of a Vector for Expressing Foreign Polypeptides at the Surface of Gram-negative Bacteria_ Gene 1988, 70:181-189.

17.

HUSEWD, SASTRY L, IVERSON S, KANG AS, ALTING-MEESM, BURTON DR, BENKOV1CSJ, LERNERRA: Generation of a Large Combinatorial Library of the ImmunoglobulJn Repertoire in Phage Lambda- Science 1989, 246:1275-1281.

21.

AGTERBERG M, ADRIAANSEH, VAN BRUGGENA, KARPERIENM, TOMMASSENJ: Outer-membrane PhoE Protein of Escherichia coli K-12 as an Exposure Vector: Possibilities and Limitations. Gene 1990, 88:37-45.

18.

SEED B, ARUFFOA. Molecular Cloning of the CD2 Antigen, the T-cell Erythrocyte Receptor, by a Rapid Immunoselection Procedure. Proc Natl Acad Sci USA 1987, 84:3365-3369.

19.

TUEe, K C, GOlD L: Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 RNA Polymerase. Science 1990, 249:505-510.

George P Smith, Division of Biological Sciences, Tucker Hall, University of Missouri, Columbia, Missouri 65211, USA,

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Surface presentation of protein epitopes using bacteriophage expression systems.

Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying...
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