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  • MINI-REVIEW

    Bacteriophage lambda display systems: developments and applications

    Jessica Nicastro & Katlyn Sheldon & Roderick A. Slavcev

    Received: 1 December 2013 /Revised: 3 January 2014 /Accepted: 4 January 2014 /Published online: 19 January 2014 # Springer-Verlag Berlin Heidelberg 2014

    Abstract Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique forma- tion of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future ap- plications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.

    Keywords Bacteriophage λ . Lytic phage display . gpD translational fusion . Gene delivery . Phage vaccine

    Introduction to bacteriophage display

    Bacteriophages (phages) are bacterial parasites that, like their animal virus counterparts, exist in a variety of morphologies and with diverse genetic architectures. Most phages are limit- ed in their host range, normally infecting only a single species of bacteria, and at many times only a subset of that species, adsorbing to specific receptors on bacterial cells that define their host range. Lytic phages such as T4 and T7 enter imme- diately into a vegetative state of reproduction upon infection, lysing the cell. In contrast, temperate phages such as bacteri- ophage Lambda (λ) “choose” between vegetative growth and a quiescent state where the phage genome is stably harbored in the host cell in a state called lysogeny. Filamentous phage,

    such as M13 and Ff do not lyse the cell in any state but rather convert the cell into a phage-producing factory, thereby compromising the host’s growth (Gulig et al. 2008).

    Bacteriophage display, or phage display, was first devel- oped by George Smith in 1985 and can be defined as the process by which a heterologous protein or peptide is expressed as an exterior fusion through the genetic fusion to a coat protein gene of the bacterial virus (phage) particle (Lindqvist 2005). This revolutionary technique, which is one of the largest innovations involving phage since their discov- ery in the early 1900s (d’Herelle and Smith 1926; Duckworth 1976), is based on the genetic fusion, where the outcome of this genotypic manipulation (Kaiser 1966) will produce a phenotypic outcome (Jestin 2008). The most well-known phage display methods are based on the usage of phage M13 and the related filamentous phage of Escherichia coli. Al- though filamentous phages are not the focus of this review, the principles of their early use govern phage display advance- ments. The most extensively used phages are derived from the E. coli Ff (filamentous) class, where the most commonly explored species include M13, fd, and f1. The basic structure of these phages consist of a circular single-stranded (ss) DNA genome that is encapsulated by a long tube comprised of thousands of copies of a single major coat protein and four additional minor capsid proteins at the tips. M13 phage has been especially useful for phage display as their genome is bound only by the major coat protein (as opposed to filling a phage head structure). Therefore, there is not a strict limit on the size of packaged DNA permitting for more opportunities for manipulation. Smith (1985) was the first to successfully demonstrate a phage capsid fusion by inserting an external gene into the phage genome of the filamentous phage fd. This construct demonstrated the efficient display of EcoRI endo- nuclease gene product on the filamentous phage fd minor coat protein pIII (Smith 1985; Smith and Huggins 1982). Based on this initial work, lytic phage, including phage T4 and T7, and

    J. Nicastro :K. Sheldon : R. A. Slavcev (*) School of Pharmacy, University of Waterloo, 200 University Avenue West Waterloo, Kitchener, ON, Canada e-mail: slavcev@uwaterloo.ca

    Appl Microbiol Biotechnol (2014) 98:2853–2866 DOI 10.1007/s00253-014-5521-1

  • temperate phage λwould eventually be successfully exploited as fusion vectors as well (Garufi et al. 2005; Kalniņa et al. 2008).

    Lytic versus filamentous phage display

    The use of filamentous phage in display technology possesses major limitations that can be overcome with lytic phage display systems. First, a tolerated fusion must have the ability to translocate across the plasma membrane and as such, many hydrophilic cytoplasmic proteins cannot be extruded as fusion proteins in filamentous phages. Second, filamentous phages are severely constrained by the size of fusions possible (Garufi et al. 2005; Mikawa et al. 1996; Sternberg and Hoess 1995). Third, filamentous phages are dependent on the viability of the host and thus cannot effectively display peptides/proteins that are toxic to the cell (Garufi et al. 2005). Due to the lytic nature of double-stranded (ds) DNA bacteriophage, the fusion of toxic proteins is no longer a concern in temperate or lytic phages as they would be repressed in the phage lysogenic state (temperate phage only) and only expressed just prior to cell lysis (Garufi et al. 2005).

    A few lytic phage candidates have been identified for phage display, including λ, T4, and T7. The C-terminus of the minor T4 fibrous structural protein fibritin that comprises the collar/whiskers complex on the neck of the phage and the C-terminal of T4 minor capsid proteins HOC and SOC toler- ate fusions for lytic phage display in T4 (Efimov et al. 1995; Ren et al. 1996). Phage T7 has been employed as a display platform for various GFP-based cytoplasmic proteins that showed poor expression (fluorescence) with more traditional filamentous display vectors (Dai et al. 2008). Santini et al. (1998) developed the first chimeric phage system in which C- terminal fusions were made to the λ major capsid protein general protein D or gpD. In this work, recombinant phages were decorated with both wild-type gpD and proteins from a hepatitis C virus (HCV) cDNA expression library. These recombinant phages were compared to two other libraries (N-terminal fusions to the pIII and pVIII capsid proteins of M13) using the cDNA expression library, as the majority of the inserts were between 100 and 300 bp in length. HCV was chosen for this study because the immune response in humans has been heavily studied (Santini et al. 1998). In order to avoid overpopulating the λ phage with gpD fusions, and improve the phage stability, they expressed a second D allele possessing an amber mutation within the gene for gpD. Here, under normal conditions gpD could not be produced by the phage unless plating was accomplished on an amber suppres- sor (SupE, SupF). This addition to the phage display process allowed for the formation of chimeric phage (Santini et al. 1998). Comparing the filamentous phage proteins, pVIII’s display was more efficient than pIII, and when pVIII was

    compared to gpD display and selection of protein fragments of limited size, the two libraries showed comparable efficien- cies. However, when larger protein fragments were tested, gpD outperformed pVIII, likely because λ does not require secretion of the fusion protein. So unlike filamentous pIII/VIII, larger recombinant proteins can fold properly with- out interfering with phage assembly while displaying higher densities. Non-homogeneous ligates such as human HCV- positive sera could also be used to identify positive phage after one or a few rounds of selection when using phage λ, which can be used to identify common immunodominant antigens in developing new vaccines.

    Gupta et al. (2003) later designed a λ phage display library that expressed fusions on the C-terminal of gpD. This library was compared to one created by fusing to an M13 phage (N- terminal fusions made utilizing major coat protein PVIII and minor coat protein PIII) where there was no degradation of displayed products. This λ display system provided a 100-fold higher display for all fragments compared to filamentous phage when tested with an antibody-binding assay. The λ system generally displayed proteins of different sizes, where the number of fusions displayed on each phage particle was 2– 3 orders of magnitude greater than that for M13. When the high-density display was applied to epitope mapping, the λ system consistently outperformedM13 with a higher enzyme- linked immunosorbant assay (ELISA) reactivity shown.

    Bacteriophage λ lytic phage display

    The icosahedral-shaped bacteriophage λ capsid is approxi- mately 60 nm in diameter with a shell thickness of 4 nm and accommodates a genome of 48.5 kb that contributes a mass of 21×106 Da (Hohn and Katsura 1977; Witkiewiczl and Schweiger 1982). The capsid is made up of two major pro- teins, gpE and gpD, where the bottom side of gpD binds to the gpE arrangement (Fig. 1) (Wurtz et al. 1976; Yang et al. 2000). The gpD, capsid protein like gpE, is incorporated at 405–420 copies on a mature phage head as strongly protruding thimble- shaped trivalent spikes. While gpE is most likely responsible for cutting the DNA into monomers and forming the initial phage prohead, gpD is necessary for phage head morphology as it stabilizes the head when filled by the full-length wild- type genome (Lankes et al. 2007; Mikawa et al. 1996). In the absence of gpD, only mutants packed with less than 82 % of the wild-type DNA can form mature phages (Imber et al. 1980; Katsura 1988; Mikawa et al. 1996; Sternberg and Hoess 1995; Sternberg and Weisberg 1977). Like e