Solid-Phase Synthesis and Combinatorial Technologies
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as mammalian retroviruses (13), baculovirus (14), and modified adenovirus (15), which are able to infect mammalian cells and are thus amplifiable in these hosts, have also been reported.
Both gram-negative and, more recently, gram-positive bacteria (1, 16, 17) have been used to display various peptide libraries that were screened to find ligands (18–20), antibodies (21, 22), and vaccines (23). Surface display on yeasts has produced the very popular yeast–two hybrid system and some of its variants; several recent papers and reviews are referenced here (24–27). These methods have been used to prepare and select polypeptides and proteins for various applications, including the identification of binding partners in protein–protein interactions where this is the technique of choice (28–31).
10.1.2 Phage Display of Polypeptide Libraries
In 1985, Smith (32) reported the insertion of foreign DNA sequences into phage genes with the resultant peptide expressed or displayed on the surface of the phage capsid. A hybrid phage displaying a foreign peptide could be isolated from wild-type phages by affinity purification using a receptor with affinity for the peptide anchored to a solid phase and washing away the unbound phage capsids. The infectivity of the phage and its ability to propagate in a suitable bacterial host were maintained by insertion of the nucleotide sequence in selected gene regions such that the isolated hybrid phage was amplified in the host to produce a large population of phages displaying the same peptide. Slightly later, Parmley and Smith applied this principle to the selection and affinity purification of different gene products (33), opening the route to several phage-displayed polypeptide libraries in 1989 (34) and 1990 (35–37). Phage display has become popular in recent years with many hundreds of papers describing a number of applications that will be discussed below after a brief description of the basic principles behind the technique.
Standard recombinant DNA techniques allow the insertion of a foreign piece of DNA into a recombinant vector, which in phage display is the wild-type phage DNA. When this phage infects its standard host, the gram-negative bacterium E. coli, the foreign DNA insert is replicated together with the phage DNA vector. Moreover, being an expression vector, the foreign insert is converted into the corresponding polypeptide sequence through translation. A peculiarity and at the same time an advantage of phage display originates from the location of the gene insert, which is introduced into the gene sequence coding for phage coat proteins and thus is expressed with them as a hybrid protein on the surface of the phage. Careful selection of the insertion loci allows the display of the foreign polypeptide as part of the phage capsid (Fig. 10.2).
Filamentous phages are typically used for phage display because of their properties, although other bacterial phages have also been used to a lesser extent (38–40). The infection of E. coli starts when one of the phage coat proteins (vide infra) connects with the pilus of the bacterial cell (Fig. 10.3, step a). The coat proteins start to dissolve and the single-stranded phage DNA (ssDNA) penetrates the cell and enters the cytoplasm while the whole virion disappears (step b). The ssDNA is replicated by the biological machinery of the host to give a double-stranded form suitable for replication
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Figure 10.2 Display of a peptide sequence on the phage surface via fusion onto phage coat proteins.
(step c) that is used by the host to produce multiple copies of the foreign ssDNA phage strand and to transcribe phage genes such as the coat proteins (step d). The progeny ssDNA strands are surrounded by the coat proteins produced and are externalized from the cytoplasm, eventually emerging from the bacterial surface as whole virions (step e, Fig. 10.3). This process, which makes on average several hundred phage particles per cell at each division cycle, continues indefinitely without significantly affecting the bacterial cell life-cycle while producing extremely large populations of phages. If the foreign DNA insert is represented by a random mixture of oligonucleotides with each sequence being recombined in a phage vector, then phage infection will amplify each ssDNA and eventually produce a population of phages each displaying a single polypeptide chain. The result of this process is a true library of displayed peptides, as shown in Fig. 10.4, where n library individuals, each displayed on x copies of phage clones, are represented.
An intriguing comparison can be made between phage display libraries and synthetic SP, pool polypeptide libraries:
•A microunit bearing multiple copies of a single library individual exists for both formats (the resin bead versus the phage virion); that is, the one-bead, one-compound concept is paralleled by the one-virion, one-compound construct.
•The location of each peptide molecule is defined on the microunit (the resin loading sites versus the phage coat protein sites) as is the loading per microunit (number of sites on a bead or on the phage surface).
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Figure 10.3 Infection and replication of phages: the whole cycle.
•Multiple copies of each library individual are easily obtained by controlling the library production steps (mix-and-split synthesis versus DNA recombination and amplification of phage populations).
•Library individuals can be screened as microunit-bound entities (on-bead screening versus on-phage screening).
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capsid. The two tips of the rod-shaped phage bear five copies of four coat proteins, two at the infecting tip (pIII and pVI, genes III and VI) and two at the other tip (pVII and pIX, genes VII and IX). The pIII proteins (200 amino acids) are responsible for the infection of bacterial hosts and, together with the abundant pVIII proteins, have been used to display polypeptides on filamentous phages. Both proteins have the N-terminus end displayed on the capsid surface, and the foreign peptide is expressed in proximity to this area. Careful selection of the insertion junctions of the foreign DNA into the genes III or VIII displays the polypeptide onto the phage surface and maintains the virion infectivity for the host and its ability to reconstruct its structure correctly while being externalized by the host. A recent report (41) validated also the use of pVII and pIX coat proteins to display, through a phagemid format, combinatorial heterodimeric arrays of antibody structures (vide infra).
The use of pIII and pVIII as supports for the polypeptide is exploitable in several ways. The virion genome may contain a single copy of either recombined gene III or VIII, displaying a peptide chain fused with five copies of pIII protein (type 3, Fig. 10.6) or around 2700 copies of pVIII (type 8, Fig. 10.6). It may contain two copies of the selected coat protein gene, one as a wild-type sequence and one as a recombinant gene, to produce a mosaic virion that displays 25–100 copies (type 88, Fig. 10.6) or even one single copy of a polypeptide chain (type 33, Fig. 10.6). Finally, the wild-type virion may be coupled with a special phage plasmid (phagemid) bearing a hybrid copy of gene III (type 3+3, Fig. 10.6) or gene VIII (type 8+8, Fig. 10.6). The presence of wild-type and hybrid phages and phagemids reduces the number of peptides displayed per phage population.
The importance of the loading per particle (from 2700 copies per phage for type VIII to even 1 copy per 100 phage virions with 3+3 constructs) is related to the activity of the displayed peptides. The presence of many peptide copies per phage particle increases the probability of spotting weakly active sequences through the additive effect of each peptide–receptor interaction. On the contrary, if only high-affinity binders are desired, a lower number of copies, or even monovalent phages, are desirable. The size of the foreign peptide sequence is also important. Small peptides may be displayed and accommodated even on the surface of a type 8 phage, but larger ones require less dense environments to maintain their flexibility as displayed sequences and to preserve the essential phage characteristics.
The structure of the phage library is determined by the sequence of the foreign DNA inserted into the coat protein phage genes. A single structure per virion is derived from the unique nature of the genetic information contained in each single phage recombined ssDNA. Library synthesis starts with the synthesis of the recombinant ssDNA strands bearing the foreign peptide coding sequences. Standard recombinant DNA techniques allow the careful control of the structure of the phage library as derived from the genetic information, and the reduced dimensions of these ssDNA chains make the production of 108–109-member libraries a reasonable task. Once prepared, the phage vectors are introduced as naked DNA into E. coli cells using electroporation (42), and replication/amplification steps immediately start following the processes depicted in Fig. 10.3. The phage population generated is freed from the E. coli cells, purified, and submitted to a selection process aimed at identifying binders for the