Research review paperPhage display: Concept, innovations, applications and future
Introduction
Fig. 1.
In phage display, exogenous (poly)peptides are expressed and presented by phage particles to bind to various target molecules. It has evolved as a powerful technology in drug discovery and in identifying and engineering polypeptides with novel functions (Paschke, 2006, Sidhu, 2000, Newton and Deutscher, 2008, Hoess, 2001, Smith & Petrenko, 1997). The foreign DNA fragment is inserted into the genome of the filamentous phage and the encoded foreign peptide is displayed as a fusion to one of the coat proteins on the surface of phage. This physical linkage of the displayed peptide on the viral particle combined with the concept of combinatorial peptide libraries have established phage display as a selection method for identifying novel ligands with desired properties. The initial concept was introduced in 1985 using filamentous bacteriophage as an expression vector (Smith, 1985). However, the use of this technique has mushroomed due to innovations introduced since then. Several reviews on the methods and applications of phage display library have been published. Here, we will briefly review the basics of the technology, modifications introduced to make it more useful and its common applications. The innovations in the basic protocol will be emphasized with the examples from the work carried out in our lab.
Our lab has been interested in the development of caloxins (specific allosteric extracellular inhibitors of plasma membrane Ca2+ pumps (Ca2+-Mg2+-ATPases, PMCA)) (Pande et al., 2005a, Pande et al., 2005b, Pande et al., 2008, Pande et al., 2006, Chaudhary et al., 2001). PMCA is a ubiquitous transmembranous protein encoded by four genes which are expressed in a tissue-dependent manner. We exploited the extracellular domains of PMCA as targets to screen the phage display random peptide libraries for allosteric inhibitors. With phage display, we have been able to obtain gene specific inhibitors of this protein. To our knowledge, these are the only known isoform specific inhibitors for any ion pumps, and thus demonstrate the power of phage display.
Section snippets
Choice of phage
The filamentous bacteriophages infect bacteria (typically E.coli is used) and in the bacterial cells, the single stranded phage genome is initially converted to double stranded replicative form that serves as a template for the production of viral coat proteins and single stranded DNA progeny (Paschke, 2006, Sidhu, 2000, Newton and Deutscher, 2008, Hoess, 2001, Smith & Petrenko, 1997). The viral coat proteins encapsulate the single stranded DNA during its extrusion from the host cell. The
Phage display libraries
The most common type of phage display libraries are the random peptide libraries. The random oligonucleotides are inserted between the coding sequence for the signal peptide and the N-terminus of the coat protein pIII. The linear random peptides varying in length from 6 to 43 amino acids have been successfully expressed as N-terminal pIII fusions (McConnell et al., 1996, Burritt et al., 1995). This allows screening of ligands for targets whose interaction site may involve residues that are
Biopanning
The most common screening method is based on enriching the phage clones with binding affinity for the target by a process called biopanning. Biopanning involves following steps: 1) target immobilization: The target molecules can be immobilized by passive adsorption to a modified 96-well polystyrene microtiter plates. The unbound target is washed off and the remaining sites in the well are blocked with unrelated proteins or nonionic detergents, 2) Phage binding: The phage display random peptide
Beyond phage display
There are enormous potentials as to what can be done beyond peptide selection by phage display. Here, examples of modification of the peptide and characterization of its binding to the target are discussed briefly. The selected peptide can be modified slightly without altering its affinity and specificity for the target. A key modification we have routinely used is to extend the length of the synthesized peptide beyond the variable sequence displayed on the phage coat protein. We have screened
Protein-protein interactions
Over 80% of cellular proteins may work in complexes with other proteins and their protein–protein interactions are regulated by several mechanisms (Berggard et al., 2007). Phage display has been used in numerous studies of protein-protein interactions (Fuh et al., 2000, Kiewitz & Wolfes, 1997, James et al., 2009, Hertveldt et al., 2009, Voss et al., 2009, Caberoy et al., 2010). Its use with combinatorial mutagenesis provides a rapid method to identify residues contributing energetically to
Conclusions and future directions
Innovations in and beyond the inceptive concept of phage display have turned it into a powerful tool. Phage display has been used to isolate novel and natural ligands for various targets and study protein-protein interactions. The selected ligands validate the target and elucidate its structure and function. Phage display has contributed to the field of immunotherapy and vaccine development. There are vast possibilities in its future. However, application aspects of phage display tools will
Acknowledgements
This work was supported by grant T 6355 from the Heart & Stroke Foundation of Ontario.
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