Lytic Phage Display System
About Lytic Display
Our lab has designed and characterized a novel 2-dimensional genetic strategy to modulate the degree of decoration of a desired peptide/protein on the surface of phage l via genetic fusion to its major capsid protein, gpD. This was done by combining various permutations of the gpD allele with controllable expression of gpD::X (fusion) during phage assembly. Our studies represent the first method and attempt to measure surface phage fluorescence (eGFP fused to gpD) on a phage by FACS and the highest degree of controllable phage decoration reported to date [Nicastro et al. 2013. Appl Microbiol Biotechnol 97: 7791-7804]. The patent describing this technology can be found here.
Tuneable lytic phage display has been applied to the generation of “superlytic nanoparticles” expressing phage-encoded S. aureus antibacterial lysin on the capsid surface (MS in preparation) as well as to target our immunogenic phage to dendritic cells via capsid fusions, where phage have also been genetically engineered to genomically encode gag-env HIV VLP therapeutic vaccine sequences. By this strategy each phage may serve as a targeted vehicle for delivery of gag-env VLP sequence for expression and VLP production in dendritic cells to generate optimized immune responses against HIV (in process).
Tuneable Lytic Phage Display
Bacteriophage, or bacterial viruses, can and have been exploited for decades for the application termed “phage display”. This strategy involves the engineered expression of a peptide(s) of interest fused to the minor and/or major coat protein of a tolerant phage. New and surprising applications of phage display are emerging, including the display of a diverse set of peptides, to clinical applications including vaccine delivery.
We have generated a novel system of phage display that is tune-able, which can prove very important in design, based on the therapeutic application. By using different suppressor strains of E. coli in combination with our patented inducible fusion production platform we can modulate surface expression of our peptide/protein of interest.
Elucidation of Rex Phenotypes
TThe Rex phenotype is defined as the inability of T4rII mutant bacteriophage to form plaques on a lawn of E. coli lysogenized by bacteriophage lambda (λ), hence excluded (Rex). Although it has been more than five decades since the discovery of the Rex phenotype by Seymour Benzer in mid 1950s, the mechanism behind this legend is still mysterious.
The T4rII exclusion (Rex) system is encoded by two genes of λ (rexA, and rexB), the expression of which is primarily regulated by the repressor gene cI from the PM promoter. The onset of Rex, triggered by T4rII infection, results in rapid membrane depolarization and a harsh cellular environment that in many ways resembles stationary phase metabolism and morphology. In addition, disruption of the RexA:RexB balance can lead to the same Rex phenotypic manifestations without infection. Despite some cell killing, infected lysogens can recover from Rex activation. Rex may thus be a mutualistic protection mechanism that protects both itself and the host cell from external infection. We have designed a system for the rapid one-step isolation of host mutations that abrogate this phenotype, in order to identify the host genes involved in Rex and elucidate the mechanism of this enigmatic exclusion system.
Find out more about the researcher: Heba Alattas