Cell Targeted Ribosome Inactivating Proteins Derived from Protein Combinatorial Libraries

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.

Department dc:contributor.department

Medical Biophysics

Year dc:date.issued

2008

Author dc:creator

• Perampalam, Subodini

Advisor dc:contributor.advisor

• Gariepy, Jean

• Protein libraries
• cytotoxicity
• anti-cancer agents
• shiga like toxin 1
• ribosome inactivating protein

Language dc:language.iso

en_ca

Handle dc:identifier.uri

http://hdl.handle.net/1807/11244

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/11244