Combinatorial Chemistry & High Throughput Screening, 2002, 5, 1-14 1 Random Sequence Libraries Displayed on Phage: Identification of Biologically Important Molecules Christopher G. Adda 1,3 , Robin F. Anders 1,3 , Leann Tilley 1,2 and Michael Foley 1,2* Department of Biochemistry, La Trobe University 1 , Bundoora, 3083, Victoria, Australia, the Cooperative Research Centre for Diagnostic Technologies 2 , and the Cooperative Research Centre for Vaccine Technology 3 Abstract: Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications. INTRODUCTION direct the display of a small foreign peptide of a defined length on the surface of a filamentous phage particle [112]. Phage displaying the foreign peptides were able to infect bacteria in the same manner as wildtype phage particles indicating that the fusion protein was still functional. A library of random peptides was then constructed and displayed on the surface of phage, at first only six amino acids in length, in which each phage particle expressed only one of 10 7 different peptides [89]. The entire phage library could be contained in a small volume of less than 1 ml and easily replicated by infecting E. coli cells. Smith screened the library using a monoclonal antibody and a technique referred to as biopanning in order to identify peptides that bound specifically to the antibody. This involved reducing the diversity of the population of peptides at each round in the biopanning process. A number of peptides were isolated, some of which contained a consensus motif that was identical to the epitope for the antibody. Another peptide was isolated that contained no such motif and showed no similarity to the epitope, yet was able to bind specifically to the antibody. Such peptides are able to mimic the conformational state of the epitope and are referred to as mimotopes as they have a different primary sequence to the epitope, yet make similar molecular contacts with the antigen-binding region of the antibody. Proteins are capable of participating in a bewildering array of interactions with other proteins and a variety of other biomolecules. It has been suggested that peptide based molecules could exhibit a massive range of possible interactions including novel associations with both organic and inorganic structures. Binding of peptides to proteins, other peptides, carbohydrates, nucleic acids and even inorganic molecules is the raw material that is likely to be exploited by the biotechnology industry seeking to develop products in medicine and biotechnology. It is not surprising therefore, that combinatorial peptide libraries are being exploited as large repertoires from which individual peptides that bind to virtually any ligand can be readily isolated. One of the simplest and most accessible means of exploring the sequence space of large peptide libraries is by phage display. Phage-display technology is a powerful molecular tool, which involves the expression of a foreign protein or peptide on the surface of bacteriophage, appended to a recombinant viral structural protein (Fig. 1). Large libraries of diverse molecular structures can be displayed on the surface of phage. Various molecules can then be used to screen for phage with a particular phenotype to obtain ligands of a desired specificity. An essential feature of phage-display technology is that it links phenotype with genotype, such that, the expression of a particular phenotype on phage is physically linked to the genetic information contained within the phage particle. The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome. Generally, enrichment of a desired specificity is based on affinity and the desired binding molecule can be rapidly replicated and its ability to interact with another molecule, easily characterized. Since the introduction of phage display, numerous applications of this technology have been described. Epitopes that are recognized by various antibodies have now been defined with no knowledge of the antigen to which they were raised [39, 76]. Epitopes recognized by antibodies that protect against infectious disease have been mimicked and the specificity of that protection, replicated [30, 75]. Peptides that bind to hormone receptors [27, 37, 135], proteins [5, 32], DNA [18] and other compounds [15, 130] have been identified using rapid screening techniques. Since its discovery, much work has been done to optimize this technology and adapt it for use in different biological situations. Various libraries, not only of peptides but also proteins, including antibody fragments, receptors and polypeptides have been designed along with a suite of affinity selection and screening techniques. George Smith pioneered phage-display technology when, in 1985, he developed a bacteriophage vector that could *Address correspondence to this author at the Department of Biochemistry, La Trobe University, Bundoora, Victoria, 3083 Australia Tel: 61-3-94792158; Fax: 61-3-9472467; e-mail: m.foley@latrobe.edu.au 1386-2073/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd.