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- W2807329315 abstract "Chemically-induced protein association is involved in key regulatory pathways throughout the body. Many natural products exert their downstream effect by the stabilisation of naturally occurring interactions, either by an allosteric effect or by the direct binding of two distinct protein partners. The targeting of these interactions by small-molecules is a relatively under developed field, with the inhibition of existing protein-protein interactions (PPIs) a more investigated target. One such method to investigate PPI inhibition is a bacterial reverse two-hybrid system (RTHS), which has been successfully coupled to a split-intein based library of cyclic peptides to identify novel inhibitors (split-intein circular ligation of peptides and proteins, SICLOPPS). Here, the existing RTHS technology was rebuilt to enable the screening of cyclic peptides for their ability to induce dimerisation of two target proteins. The well-studied mTOR-FKBP12 interaction, with heterodimerisation induced by the natural product rapamycin, was used to build and validate a novel two-hybrid system. This utilises the tetracycline repressor to reverse the transcriptional control from the RTHS, leading to a selectable phenotype upon dimerisation. Rapamycin addition successfully results in the association of these target proteins, leading to the downstream expression of two essential reporter genes and a fluorescent protein. This rapamycin-dependent interaction can be identified both by an agarbased growth assay, and a liquid media-based fluorescent assay. A large number of SICLOPPS libraries were screened with little success at replicating the action of brapamycin. The SICLOPPS technology was optimised and expanded, enabling an improved screening platform for the future." @default.
- W2807329315 created "2018-06-13" @default.
- W2807329315 creator A5084612369 @default.
- W2807329315 date "2017-11-01" @default.
- W2807329315 modified "2023-09-22" @default.
- W2807329315 title "Development of a novel two-hybrid system for the identification of cyclic peptide induced protein-protein association" @default.
- W2807329315 hasPublicationYear "2017" @default.
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