The conditional protein splicing of alpha-sarcin: a model for inducible assembly of protein toxins in vivo.

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dc.contributor.author Alford, Spencer C.
dc.date.accessioned 2007-08-09T21:28:14Z
dc.date.available 2007-08-09T21:28:14Z
dc.date.copyright 2007 en_US
dc.date.issued 2007-08-09T21:28:14Z
dc.identifier.uri http://hdl.handle.net/1828/186
dc.description.abstract Conditional protein splicing (CPS) is an intein-mediated post-translational modification. Inteins are intervening protein elements that autocatalytically excise themselves from precursor proteins to ligate flanking protein sequences, called exteins, with a native peptide bond. Artificially split inteins can mediate the same process by splicing proteins in trans, when intermolecular reconstitution of split intein fragments occurs. An established CPS model utilizes an artificially split Saccharomyces cerevisiae intein, called VMA. In this model, VMA intein fragments are fused to the heterodimerization domains, FKBP and FRB, which selectively form a complex with the immunosuppressive drug, rapamycin. Treatment with rapamycin, therefore, heterodimerizes FKBP and FRB, and triggers trans-splicing activity by proximity association of intein fragments. Here, we engineered a CPS model to assemble inert fragments of the potent fungal ribotoxin, alpha (α)-sarcin, in vivo. Using this model, we demonstrate rapamycin-dependent protein splicing of α-sarcin fragments and a corresponding induction of cytotoxicity in HeLa cells. We further show that permissive extein context and incubation temperature are critical factors regulating the splicing of active target proteins. Ultimately, this technology could have potential applications in the fields of developmental biology and anti-tumour therapy. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Intein en_US
dc.subject Sarcin en_US
dc.subject protein splicing en_US
dc.subject.lcsh UVic Subject Index::Sciences and Engineering::Biology::Molecular biology en_US
dc.title The conditional protein splicing of alpha-sarcin: a model for inducible assembly of protein toxins in vivo. en_US
dc.type Thesis en_US
dc.contributor.supervisor Howard, Perry
dc.degree.department Dept. of Biochemistry and Microbiology en_US
dc.degree.level Master of Science M.Sc. en_US

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