Engineering M13 for phage display
Introduction
Phage display is a powerful method for selecting and engineering polypeptides with desired binding specificities [1], [2]. The method relies on the fact that if gene fragments encoding polypeptides are fused to M13 coat protein genes, these ‘fusion genes’ can be incorporated in bacteriophage particles that also display the heterologous proteins on their surfaces [3]. In this way, a physical linkage is established between phenotype and genotype.
Using simple molecular biology techniques, large and diverse phage-displayed polypeptide libraries can be generated. Phage displaying polypeptides with a desired binding specificity can be selected from library pools by binding to an immobilized ligand, and the sequences of selected polypeptides can be deduced from the sequence of the encapsulated DNA. As with any combinatorial method, the success of phage display depends on the size and quality of the initial library. The need for large libraries has been widely appreciated, and current optimized methods enable the facile construction of phage display libraries containing >1011 unique DNA sequences [4].
However, the utility of a phage-displayed library also depends on the display quality, because a particular library member is only selectable if the DNA-encoded polypeptide is efficiently displayed on the phage surface. The levels of display for different polypeptides vary greatly and depend on both the length and sequence of the displayed polypeptide [5], [6]. Hundreds or even thousands of copies of small peptides (<8 residues) can be displayed per phage particle [7], [8], while large proteins (>100 residues) are generally displayed at monovalent levels of one copy or less per phage particle [9], [10], [11], [12]. Furthermore, the utility of phage libraries for particular applications can also depend on whether the libraries are fused to the N or C terminus of the phage coat protein [13], [14], [15].
This review summarizes progress in engineering M13 coat proteins for improved performance as phage display scaffolds. These studies have expanded the utility of the phage display technology by providing a greater variety of N- and C-terminal display scaffolds [13], [14], [15], [16]. Also, it has been shown that mutant coat proteins can be specifically selected to provide increased levels of heterologous protein display, thus improving the quality of phage-displayed libraries and enabling the display of proteins that could not be displayed with wild-type coat proteins [12], [17]. Finally, it has been demonstrated that phage display scaffolds need not be restricted to the natural coat proteins; completely artificial coat proteins have been designed and selected de novo for the purposes of phage display [18]. These studies reveal that M13 phage assembly is a promiscuous process, and the phage coat can be engineered for improved phage display.
Section snippets
M13 bacteriophage structure
M13 is an Escherichia coli-specific filamentous bacteriophage ≈1 μm in length but less than 10 nm in diameter. The particle consists of a single-stranded DNA core surrounded by a proteinaceous coat [19]. The coat contains five different proteins (Fig. 1), but the vast majority consists of several thousand copies of the gene-8 major coat protein (protein-8, P8) which covers the length of the particle. The four minor coat proteins are present at about 5 copies per particle; protein-7 and
M13 bacteriophage assembly
The process of M13 bacteriophage assembly has been reviewed in detail elsewhere [27], [28], and an abbreviated description is presented here. The M13 genome contains 11 genes; five of these genes encode the coat proteins described above while the others encode proteins necessary for viral replication and assembly. M13 replication is initiated when P3 binds to the F pilus on the surface of an E. coli cell, and then facilitates the translocation of the viral DNA into the E. coli cytoplasm. Inside
Platforms for phage display
Polypeptides fused to M13 coat proteins will be displayed on the surface of the phage particle, provided the fusion protein can pass successfully through the assembly apparatus and integrate into the assembling phage without significantly effecting the viability of the phage particle itself. The earliest phage display systems relied on fusions to the N terminus of either P3 or P8 in the viral genome [8], [29], [30], [31], [32], [33], but their utility was limited because polypeptides that
Engineering the M13 coat for improved phage display
P8 is a 50-residue α-helix that can be divided into three distinct functional regions (Fig. 4) [19]. The first 20 residues comprise an amphipathic helix with a hydrophobic face that is buried against the phage coat and a hydrophilic face that is exposed on the outside of the phage particle. This region is followed by a 19-residue hydrophobic domain that spans the E. coli inner membrane prior to phage assembly; in the phage particle, adjacent hydrophobic domains pack against each other and
Artificial M13 coat proteins
The fact that highly mutated P8 molecules can be incorporated into the M13 coat suggested that it may be possible to engineer completely synthetic coat proteins using simple design and selection principles [18]. The P8 α-helix was viewed as a scaffold that presents the clusters of side chains that are the actual recognition elements guiding incorporation into the phage coat. In this view, it seemed reasonable that the structure of the coat protein main chain may not be crucial for function, and
Conclusions
Phage display has proven to be a powerful technology for selecting and engineering novel protein functions (reviewed in [4], [40], [41]). The success of phage display depends not only on the diversity of the library at the DNA level, but also on the efficiency with which the encoded proteins are displayed on the phage surface. In hybrid phage display systems, the fusion protein is an additional coat component that has little effect on phage stability and viability, and thus, hybrid phage have
Acknowledgements
I thank Charles Eigenbrot and David Wood for assistance with figures.
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