Abstract
Since its introduction in 1985, phage display technology has been successfully used in projects aimed at deciphering biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical molecular cloning and expression system has also been exploited for generating large combinatorial libraries of small peptides and protein domains exposed on its capsid. More recently, lambda display has been consistently and successfully employed for domain mapping, antigen discovery and protein interaction studies or, more generally, in functional genomics. We show here the results obtained by the use of large libraries of cDNA and genomic DNA for the molecular dissection of the human B-cell response against complex pathogens, including protozoan parasites, bacteria and viruses. Moreover, by reviewing the experimental work performed in recent investigations we illustrate the potential of lambda display in the diagnostics field and for identifying antigens useful as targets for vaccine development.
Highlights
Since its introduction in 1985, phage display technology has been successfully used in projects aimed at deciphering biological processes and isolating molecules of practical value in several applications
The results obtained by the use of lambda display for antigen discovery are summarized in Table 1 and some valuable applications are further discussed below using the T. gondii project as a model system
The results showed that DNA immunization with microneme fragments elicited effective protection in mice (84% reduction in brain-cyst burden), strongly indicating that a combination of these antigenic regions should be considered in the design of vaccines against toxoplasmosis
Summary
Display technologies allow the exploration of large repertoires of biological molecules by the use of efficient selection and rapid characterization procedures. Nucleotide sequence repertoires such as mRNAs, cDNAs, genomic DNA fragments, and synthetic oligonucleotides are cloned into bacteriophage genomes with a specific phenotype/genotype linkage: each virus contains the genetic information for the ectopic element displayed on its capsid In such a way the displayed recombinant molecule can interact with the corresponding target allowing the isolation of specific phage clones from pools of billions of distinct recombinant viruses. Recent alternative display systems exploited three lytic bacteriophages, characterized by very different life cycles, but sharing the common property of being assembled in the cytoplasm and released by cell lysis: lambda [32,33,34,35,36] T7 [37] and T4 [38,39] In these bacteriophages, the display of fusion proteins does not depend on their ability of being translocated across the bacterial membrane. The filamentous phage display is suited for the expression of secreted proteins, while the lambda phage and other lytic bacteriophages (T7 and T4) are excellent for the display of cytoplasmic proteins
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