Chapter 1 Introduction to phage biology and phage display Marjorie Russel The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Henry B.Lowman Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Tim Clackson ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, MA 02139, USA. 1 Introduction Display of peptides and proteins on ®lamentous phageÐphage displayÐis an in vitro selection technique that enables polypeptides with desired properties to be extracted from a large collection of variants. A gene of interest is fused to that of a phage coat protein, resulting in phage particles that display the encoded protein and contain its gene, providing a direct link between phenotype and genotype. This allows phage libraries to be subjected to a selection step (e.g. af®nity chromatography), and recovered clones to be identi®ed by sequencing and re-grown for further rounds of selection. Since the initial description of the approach by Smith (1), it has become established as a powerful method for identifying polypeptides with novel properties, and altering the properties of existing ones (for reviews, see 2±5). Filamentous phages are ideal in many ways for use as cloning vehicles and for display in particular. The genome is small and tolerates insertions into non- essential regions; cloning and library construction are facilitated by the ability to isolate both single- and double-stranded DNA (ssDNA and dsDNA), and by the availability of simple plasmid-based vectors; coat proteins can be modi®ed with retention of infectivity; phage can accumulate to high titers since their pro- duction does not kill cells; and phage particles are stable to a broad range of potential selection conditions. This chapter is intended to provide the background needed to initiate a phage display project. In the ®rst portion (Sections 2 and 3), the life cycle, genetics, and 1 MARJORIE RUSSEL ET AL. structural biology of ®lamentous phages are summarized, with a focus on aspects that are relevant to phage display. We then go on to describe general considera- tions to be made when approaching a new phage display project, including choice of display format, experimental design, and common pitfalls (Sections 4±6). Finally, summaries of commercial sources of phage display vectors, kits, and alternative display systems are provided for those cases in which such reagents can provide a head-start for investigators (Sections 7 and 8). Cross-references are provided to later chapters in the book that provide detailed procedures. 2 Biology of filamentous phage 2.1 Introduction Filamentous phages constitute a large family of bacterial viruses that infect many gram-negative bacteria. Their de®ning characteristic is a circular, ssDNA genome encased in a long, somewhat ¯exible tube composed of thousands of copies of a single major coat protein, with a few minor proteins at the tips. The genome is smallÐa dozen or fewer closely packed genes and an intergenic (IG) region that contains sequences necessary for DNA replication and encapsidation. Unlike most bacterial viruses, ®lamentous phages are produced and secreted from infected bacteria without cell killing or lysis (Figure 1). Readers are referred to several excellent reviews on ®lamentous phage (6±9) for more comprehensive information and citations of the primary literature than are given here. Most information about ®lamentous phages derives from those that infect E. coli: f1/M13/fd, and to a lesser extent IKe. These phages are characterized by a ®vefold rotation axis combined with a twofold screw axis. Phages f1, M13, and fd are those that have been used for display. Their genomes are more than 98% identical and their gene products interchangeable, and the phages are usually referred to collectively, as Ff phages. Unless speci®ed, the properties of ®lamen- tous phage described below refer to them together. 2.2 Structure of the phage particle Filamentous phages have a ®xed diameter of about 6.5 nm and a length deter- mined by the size of their genome. The 6400-nucleotide ssDNA of Ff is encapsi- dated in a 930 nm particle, while a 221-nucleotide ``microphage'' variant is 50 nm long (10). Cloning DNA into a nonessential region of the genome can create longer phage, although the longer they are, the more sensitive the particles are to breakage (e.g. from vortexing). Phage particles are composed of ®ve coat proteins (Figure 2). The hollow tube that surrounds the ssDNA is composed of several thousand copies of the 50-residue major coat protein, pVIII, oriented at a 20 angle from the particle axis and overlapped like ®sh scales to form a right-handed helix (8). The ®lament is held together by interactions between the hydrophobic midsections of adjacent subunits. Except for ®ve surface-exposed N-terminal residues, pVIII forms a single, continuous -helix. The four positively charged residues near 2 INTRODUCTION TO PHAGE BIOLOGY AND PHAGE DISPLAY Ff bacteriophage E.coli PS ssDNA Gene TolA expression F pilus dsDNA (RF) pIII + pVI pVIII pI/pXI + pIV pV dimers pVII + pIX Thioredoxin Figure 1 Life cycle of ®lamentous phage f1 (M13/fd).Sequential binding of pIII to the tip of the F-pilus and then the host Tol protein complex results in depolymerization of the phage coat proteins, their deposition in the cytoplasmic membrane (where they are available for re- utilization), and entry of the ssDNA into the cytoplasm.The ssDNA is converted by host enzymes to a double-stranded RF, the template for phage gene expression.Progeny ssDNA, coated by pV dimers (except for the packaging sequence hairpin (PS) that protrudes from one end), is the precursor of the virion.A multimeric complex that spans both membranesÐ composed of pI, pXI, pIV, and the cytoplasmic host protein thioredoxinÐmediates conversion of the pV±ssDNA complex to virions and secretion of virions from the cell.This process involves removal of pV dimers and their replacement by the ®ve coat proteins that transiently reside in the cytoplasmic membrane. the C-terminus of pVIII are at the inner surface of the tube and interact with phosphates of the viral ssDNA. The ends of the particle are distinguishable in electron micrographs. The blunt end contains several (3±5) copies each of pVII and pIX, two of the smallest ribosomally translated proteins known (33 and 32 residues, respectively). Neither their structure nor disposition in the particle is known. However, immunological evidence indicates that at least some of pIX is exposed (11) and antibody variable regions have been successfully displayed on the amino termini of pVII and pIX (12). Phage assembly begins at the pVII±pIX end, and in the absence of either protein, no particle is formed. The pointed end of the particle contains about ®ve copies each of pIII and pVI, both of which are needed in order for the phage to detach from the cell mem- brane; pVI is degraded in cells that lack pIII, which suggests that these proteins 3 MARJORIE RUSSEL ET AL. II/X V VII IX VIII III VI I/IX IV Replication Virion Assembly/export Figure 2 Filamentous phage f1 (M13/fd) genes and gene products. Gene II encodes pII, which binds in the IG region (located between genes IV and II/X; not shown) of dsDNA and makes a nick in the strand, initiating replication by host proteins.pX is required later in infection for the switch to ssDNA accumulation. Gene V encodes the ssDNA binding protein pV. Genes VII and IX encode two small proteins located at the tip of the virus that is ®rst to emerge from the cell during assembly. Gene VIII encodes the major coat protein, and genes III and VI encode pIII and pVI, which are located at the end of the virion and mediate termination of assembly, release of the virion, and infection. Gene I encodes two required cytoplasmic membrane proteins, pI and pXI, and gene IV encodes pIV, a multimeric outer membrane channel through which the phage exits the bacterium.Note that the genome is in fact circular, but is shown in a linear presentation here for clarity. assemble in the cell membrane before their incorporation into phage particles (13). They can be isolated from phage as a complex (14). The disposition of pVI in the particle is not known, but pVI with fusions to the C-terminus can be incor- porated into phage, suggesting that this portion of the 112-residue pVI may be surface exposed (13). More is known about the 406-residue pIII, the most commonly used coat protein for display (Figure 3). Its N-terminal domain, which is necessary for phage infectivity, is surface exposed and forms the small ``knobs'' that can often be seen to emanate from the pointed end of the particle in electron micro- graphs. Three pIII domains have been de®ned, the two N-terminal of which (N1 and N2) are believed to interact intramolecularly, based on crystallographic analysis (16, 17). The three domains are separated by two long, presumably ¯exible linkers characterized by repeats of a glycine-rich sequence. The ®nal 132 residues within the C-terminal CT domain are necessary and suf®cient for pIII to be incorporated into the phage particle and to mediate termination of assembly and release of phage from the cell; this domain is likely to be buried within the particle (13). The single-stranded phage genome is oriented within the phage particle. Its orientation is determined by the packaging signal (PS), located in the non-coding IG region of the genome. The PS, an imperfect but extremely stable hairpin, is positioned at the pVII±pIX end of the particle and is necessary and suf®cient for ef®cient encapsidation of circular ssDNA into phage particles.