Identifying Natural Proteinprotein Interactions

Genome sequencing projects have provided vast databases at the DNA level and have identified thousands of open reading frames (Venter et al. 2001). Consequently, the demand for technologies that enable the functional analysis of gene products is at a premium. Furthermore, the identification and subsequent characterization of protein-protein interactions have become central to drug discovery and biotechnology. Because phage display provides a physical linkage between proteins and encoding DNA, the technology has proven useful for the screening of protein interaction pairs. Indeed, the very first report of phage display suggested that the method could be used to search DNA libraries for interactions at the protein level (Smith 1985).

Highly diverse display libraries have been constructed by fusing either cDNA (Crameri and Suter 1993; Crameri et al. 1994; Jespers et al. 1995; Dunn 1996; Rhyner et al. 2002) or genomic DNA fragments (Palzkill et al. 1998; Jacobsson and Frykberg 2001) to the genes encoding for pIII or pVIII. Conventional fusions to the N-termini of phage coat proteins are suboptimal for these applications because display is impaired by the early termination of protein translation due to the presence of stop codons at the ends of open reading frames (Crameri et al. 1996; Crameri and Kodzius 2001; Walter et al. 2001). However, it is possible to functionally display polypeptides fused to the C-termini of phage coat proteins (Kang et al. 1991; Jespers et al. 1995; Fuh and Sidhu 2000; Gao et al. 2002). Although not widely used, C-terminal fusions can provide functional display of cDNA libraries despite the presence of stop codons. A more widely used indirect strategy that also overcomes the problem of stop codons relies on heterodimer leucine zippers fused to the N- or C-termini of phage proteins or displayed proteins, respectively (Crameri and Suter 1993). This method has been successfully applied to the analysis of IgE antigens from various allergenic sources (Crameri and Kodzius 2001; Rhyner et al. 2002).

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