The Emerging Impact of Phage Display Technology in Thrombosis and Haemostasis

Introducation

The phage display technology represents a powerful tool for protein and drug design because vast numbers of amino acid sequences can be rapidly explored. This review describes the origins of phage libraries, their evolution and more recent advances, including examples in the area of thrombosis and haemostasis, where the phage display approach has just begun to be used with great success.

Phage display uses filamentous phages (E. coli specific phages with a filamentous shape that contain a single stranded closed circular molecule of DNA), such as M13 or fd, as vehicles for displaying foreign peptides or proteins on their surface. This is carried out by fusing the coding sequence (DNA) for the peptide or protein to the amino (N)-terminus of either full-length phage minor coat protein III (cpIII), or to phage major coat protein VIII (cpVIII), to carboxy (C)-terminal domain of cpIII or, more recently, by fusion to the C-terminus of full-length phage minor coat protein VI (cpVI) (Fig. 1). Expression of the fusion protein and its subsequent incorporation into the mature phage particle results in the foreign peptide or protein being presented on the phage surface. Thus, the linkage of each peptide or protein to its encoding genetic material [contained as part of the single-stranded viral DNA (1, 2)] represents a great advantage over conventional cloning methods.

The phage display approach was first used by Smith in 1985 (3), who expressed a library of peptide sequences at the N-terminus of cpIII (3-5). This insertion allowed phage assembly and display of the peptide on the phage surface, without affecting the phage infectivity significantly. The linkage of genotype and phenotype in the phage library allowed facile isolation of clones of specific interest from pools of millions of clones by successive rounds of phage affinity selection on surfaces coated with a ligand (panning) followed by phage amplification by infecting male E. coli (Fig. 2).

The foreign peptides or proteins were displayed, initially, on every copy of the coat protein, but only short peptides can be displayed in this way without altering the phage infectivity. Two systems have been developed to solve this problem. One incorporates a second native gene III or VIII in the phage genome giving a mixture of native and recombinant coat protein incorporation on the phage. The second system provides the recombinant cpIII, cpVIII or cpVI gene on a phagemid (a plasmid containing the origin of replication of filamentous phage). Phagemids can be packaged into phage particles by superinfection with a helper phage. The fusion protein is incorporated onto the surface coat, along with copies of the native coat protein encoded by the helper phage. The result is a mixture of wild-type helper phage and recombinant phagemid particles, but due to a defective origin of replication the helper phage is poorly packaged to provide minimal competition with the phagemids (6). Phages that display both native coat protein and fusion protein are infective. Therefore, the display systems can be either multivalent or monovalent. Multivalent systems make use of gene III phage constructs or gene VIII phage or phagemid constructs and give a high number of foreign domains displayed on their surface (7). Monovalent systems utilize gene III or gene VI phagemid constructs, which have a low number of foreign domains displayed on their surface, usually a single copy. Consequently, monovalent systems distinguish between low affinity and high affinity clones in panning assays (1, 8).

• References

• 1 Barbas III CF, Kang AS, Lerner RA, Benkovic SJ. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci USA 1991; 88: 7978-82.
• 2 Kang AS, Barbas III 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: 4363-6.
• 3 Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the surface of the virion. Science 1985; 228: 1315-7.
• 4 Parmley SE, Smith GP. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 1988; 73: 305-18.
• 5 Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990; 249: 386-90.
• 6 Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol 1987; 153: 3-11.
• 7 Barbas III CF. Recent advances in phage display. Curr Opin Biotechnol 1993; 4: 526-30.
• 8 Smith GP. Surface presentation of protein epitopes using bacteriophage expression systems. Curr Opin Biotechnol 1991; 2: 668-73.
• 9 Hoess RH. Phage display of peptides and protein domains. Curr Opin Struct Biol 1993; 3: 572-9.
• 10 Cesareni G. Peptide display on filamentous phage capsids; a new powerful tool to study protein-ligand interaction. FEBS Lett 1992; 307: 66-70.
• 11 Smith GP, Scott JK. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol 1993; 217: 228-57.
• 12 Cortese R, Monaci P, Luzzago A, Santini C, Bartoli F, Cortese I, Fortugno P, Galfre G, Nicosia A, Felici F. Selection of biologically active peptides by phage display of random peptide libraries. Curr Opin Biotechnol 1996; 7: 616-21.
• 13 Stephen CW, Lane DP. Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library. J Mol Biol 1992; 225: 577-83.
• 14 Geysen HM, Rodda SJ, Mason TJ. A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol Immunol 1986; 23: 709-15.
• 15 Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 1990; 87: 6378-82.
• 16 Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries: a source of specific protein binding molecules. Science 1990; 249: 404-6.
• 17 Oldenburg KR, Loganathan D, Goldstein IJ, Schultz PG, Gallop MA. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc Natl Acad Sci USA 1992; 89: 5393-7.
• 18 Scott JK, Loganathan D, Easley RB, Gong X, Goldstein IJ. A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc Natl Acad Sci USA 1992; 89: 5398-402.
• 19 Hoess RH, Brinkmann U, Handel T, Pastan I. Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3. Gene 1993; 128: 43-9.
• 20 Christian RB, Zuckerman RN, Kerr JM, Wang L, Malcom BA. Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage. J Mol Biol 1992; 227: 711-8.
• 21 Bottger V, Lane EB. A monoclonal antibody epitope on keratin 8 identified using a phage peptide library. J Mol Biol 1994; 235: 61-7.
• 22 Du Plessis DH, Wang L-F, Jordaan FA, Eaton BT. Fine mapping of a continuous epitope on VP7 of bluetongue virus using overlapping synthetic peptides and a random epitope library. Virology 1994; 198: 346-9.
• 23 Geysen HM, Rodda SJ, Mason TJ, Tribbick G, Schoffs PG. Strategies for epitope analysis using peptide synthesis. J Immunol Methods 1987; 102: 259-74.
• 24 Horn IR, van den Berg BMM, Moestrup SK. Van Zonneveld A-J, Epitope mapping of an anti-α₂MR/LRP mAB using a bacteriophage displayed α₂MR/LRP random fragment library. In: Schlossman S, editor. Leucocyte Typing V, Oxford University Press; 1995: 975-8.
• 25 VanZonneveld A-J, van der Berg BMM, van Meijer M, Pannekoek H. Identification of functional interactions sites on proteins using bacteriophage-displayed random epitope libraries. Gene 1995; 167: 49-52.
• 26 Arza B, Felez J, Lopez-Alemany R, Miles LA, Munoz-Canoves P. Identification of an epitope of α-enolase (a candidate plasminogen receptor) by phage display. Thromb Haemost 1997; 78: 1097-103.
• 27 Liang OD, Rosenblatt S, Chhatwal GS, Preissner KT. Identification of novel heparin-binding domains of vitronectin. FEBS Lett 1997; 407: 169-72.
• 28 Nagesha HS, Yu M, Wang L-F. Application of linker-ligation-PCR for construction of phage display epitope libraries. J Virol Methods 1996; 60: 147-54.
• 29 Luzzago A, Felici F, Tramontano A, Pessi A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides. I. Epitope mapping of human H ferritin using a phage library of contrained peptides. Gene 1993; 128: 51-7.
• 30 Felici F, Luzzago A, Folgori A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides. II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries. Gene 1993; 128: 21-7.
• 31 O’Neil KT, Hoess RH, Jackson SA, Ramachandran NS, Mousa SA, DeGrado WF. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins 1992; 14: 509-15.
• 32 McLafferty MA, Kent RB, Ladner RC, Markland W. M13 bacteriophage displaying disulfide-constrained microproteins. Gene 1993; 128: 29-36.
• 33 Barbas III CF, Bain JD, Hoekstra DM, Lerner RA. Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci USA 1992; 89: 4457-61.