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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/11244

Title: Cell Targeted Ribosome Inactivating Proteins Derived from Protein Combinatorial Libraries
Authors: Perampalam, Subodini
Advisor: Gariepy, Jean
Department: Medical Biophysics
Keywords: Protein libraries
cytotoxicity
anti-cancer agents
shiga like toxin 1
ribosome inactivating protein
Issue Date: 1-Aug-2008
Abstract: Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.
URI: http://hdl.handle.net/1807/11244
Appears in Collections:Doctoral
Department of Medical Biophysics - Doctoral theses

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