Lab Members: Melissa Cheung,Eric Huang
The design and use of combinatorial libraries is one of the most dynamic and fastest moving fields in molecular biology. We are interested in developing targeted anti-cancer agents using various combinatorial strategies. The first strategy involves using a member of the ribosome inactivating protein family, Shiga Like Toxin-1 (SLT-1), to profile the surface of tumor cells for markers that are internalized by cells¹. We are using the A1 chain of SLT-1 which contains the catalytic activity as a protein template to engineer a receptor binding function.
The second strategy involves the development, characterization and functionalization of aptamers against a variety of tumor markers (Please refer to tumour biology for more information).
Figure 1A. Linear representation of a combinatorial SLT-1A library design. The library was created by inserting a randomized 7-residue peptide between residues H245 and A246 of the cytotoxic A domain of SLT-1. The B domain of SLT-1 is then removed and the purified toxin variants are screened to determine variants which selectively kill human cancer cells.
Figure 1B. Space-filling model of a SLT-1A chain displaying a heptapeptide insert (blue) between H245 and A246. Residues implicated in the catalytic activity of the A chain are shown in purple.
Figure 2. Flow diagram of how we screen our combinatorial protein libraries.
Figure 3. Representative cell survival assay diagram for 94 toxin variants purified and seeded onto cancer cells.
- LaPointe P, Wei X, and Gariépy J. (2005). A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen. J Biol Chem 280(24); 23310-82.
- Bray MR, Bisland S, Perampalam S, Lim WM, Gariépy J. (2001). Probing the surface of eukaryotic cells using combinatorial toxin libraries. Curr Biol. 11(9);697-701.