Because of its robustness and ease of use, phage display has been the method most widely used in the past decade. This review therefore focuses on libraries using this selection principle. For an overview of available strategies and protocols, see McCafferty et al. (31) and Kontermann and Dübel (32). Display systems that require the insertion of antibody genes into the phage genome have been developed for phage T7 (33), phage Lambda (7,34,35), and the Ff class (genus inovirus) of the filamentous phage f1, fd, and M13 (10). Being well established for peptide display, the phage T7 is not well suited for antibody phage display because protein assembly takes place in the reducing milieu of the cytoplasm, thus leaving most antibodies unfolded (33). In contrast, the oxidizing milieu of the bacterial periplasm allows antigen binding fragments of antibody heavy and light chain to be folded and assembled properly (36). The Ff class nonlytic bacteriophage are assembled in this cell compartment and allow the production of phage without killing the host cell. This is a major advantage compared with the lytic Lambda phage (7). In addition, filamentous phage allow the production of soluble proteins by introducing an amber stop codon between the antibody gene and gene III. In an E. coli supE suppressor strain, the fusion proteins will be produced, whereas soluble antibodies are made in a nonsuppressor strain (37,38). As a result the members of the Ff class are the phage of choice for antibody phage display.
To achieve surface display, five of the M13 coat proteins have been used in fusion to foreign protein fragments. In the most widespread system the antibody is coupled to the N-terminus or second domain of the minor coat protein pIII (11,12,14). The naive function of the three to five copies of the pIII, in particular their N-terminal domain, is to provide binding of the phage to the f-pili of E. coli to initiate infection (39). The major coat protein (pVIII) has been used as an alternative fusion partner, with only very few successes reported in the past decade (40). This fusion technique is more useful for the display of short peptides (41,42). Fusions to pVI have been tried, but not yet with antibodies (43). pVII and pIX were used in combination by using the variable part of light chain domain to pIX and the variable part of heavy chain domain to pVII, which allowed the expression of a fragment variable (Fv) on the phage surface. This particular method offers the potential for heterodimeric display (44). However, the fusion with pill remains the most widely used system for phage display and is the only system of practical relevance so far.
Two different systems have been developed for the expression of the antibody/pill fusion proteins. First, the fusion gene can be inserted directly into the phage genome substituting the wildtype pIII (10). Second, the fusion gene can be provided on a separate plasmid with an autonomous replication signal, promoter, resistence marker and phage morphogenetic signal, allowing this "phagemid" to be packaged into assembled phage particles. A helper phage, usually M13K07, is necessary for the production of the antibody phage to complement the phage genes not encoded on the plasmid. Because of its mutant origin, the M13K07 helper phage genome is not efficiently packaged during antibody phage assembly when compared with the phagemid (45), thus increasing the selection of the phagemid of interest during panning.
What are the practical differences of the vectors described so far? In the system using direct insertion into the phage genome, every pIII protein on a phage carries an antibody fragment. This is of a particular advantage in the first round of panning, where the desired binder is diluted by millions of phage with unwanted specificity. The oligovalency of these phage improves the chances of a specific binder being enriched because of the improved binding provided by the avidity effect. This advantage, however, has to be weighed against a number of disadvantages. The transformation efficiency of phagemids is two to three orders of magnitude better than phage vectors, thus facilitating the generation of large libraries in phagemids. Second, the additional protein domains fused to pill may reduce the function of pIII during reinfection. In a phagemid system, the vast majority of the pill assembled into phage are wildtype (wt) proteins, thus providing normal pilus binding. This may explain why only two "single-pot" antibody libraries (38,46) have ever been made using phage vectors. In phagemid systems, by contrast, both replication and foreign fusion protein expression are independent from the phage genome. Because the propagation of the phagemid occurs in the absence of helper phage, there is no selection pressure from this end. The fusion protein can be produced in adjustable quantities by the use of the amber stop/suppressor system for switching expression to antibody (Ab) fragments without a pIII domain Finally, despite not usually being derived from highest copy plasmids, the dsDNA of phagemids is more easy to handle than phage DNA, facilitating both cloning and analysis. Therefore, most pIII display systems use the phagemid approach. There is, however, a disadvantage that originates from the two independent sources for the pIII during phage packaging. During assembly, the wt pIII of the phage are inserted into the phage particles with much higher rate than the pIII fusion protein. As a result, the vast majority of resulting phage particles carry no antibody fragments at all. The few antibody phages in these mixtures are therefore mainly monovalent, with phage carring two or more antibodies being extremely rare. This allows the selection of antibodies with high monovalent affinity, since avidity effects which would decrease the dissociation rate from the panning antigen can be avoided. In the first panning round, however, when a few binders have to be fished out of a huge excess of unwanted phage, the fact that only a few percent of the phage carry antibodies hampers the efficiency of the system (11,12,14,46-48). Recently, this problem has been solved by using a helper phage (Hyperphage), which avoids the need to introduce wt pIII into the packaging process, thus leaving the phagemid as the sole source of pIII and therefore offering multivalent display for phagemid vectors as well. This method improves Ab display by two orders of magnitude and vastly improves panning efficiency (49). Multivalent display has however been achieved by integrating two amber stop codons into the gIII gene of the helper phage genome. This allows the production of a functional helper phage (ex-phage) in an E. coli suppressor strain. In the associated phagemid pIGT3, the Ab/pIII fusion occurs without an amber stop codon and the Ab phage is produced in an E. coli nonsuppressor strain (50). However, the deletion of the amber stop codon in the phagemid makes it imperative that the antibody gene is subcloned or that a protease is used to produce soluble antibodies. This is in contrast to the Hyperphage system where the amber stop/supressor system can be used for switching expression to Ab fragments without a pIII domain.
An elegant application of phage display is selective infective ph-age technology. Here, antibodies are fused to the C-terminal domain of pIII by cloning into the phage genome; therefore, every pIII carries an antibody and deletion of the pIII aminoterminal region makes the phage noninfective. Antigen is then fused to the C-termi-nal end of separately produced soluble pIII N-terminal domain. The functional, f-pili binding pIII is reconstituted when the antibody phage binds to the antigen, allowing only the correct antibody ph-age to infect E. coli and be propagated (51). However, because of the fast kinetics of pIII/pilin interactions and very low concentrations of the three reaction partners if not co-expressed in the same cell, this method does not lend itself to the rapid panning of larger libraries.
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