Expression, purification and structure determination of disulfide-rich cone snail toxins

Main area:
Target group:Biochemistry, Biology, Molecular Biomedicine
Educational level:Masters
Project description:
Predatory marine cone snails are amazing creatures that kill their prey – fish, worms or other snails – by injecting them with a deadly mixture of hundreds of different disulfide-rich neuropeptides referred to as conotoxins. Conotoxins selectively target specific subtypes of receptors or ion channels throughout the nervous system, a characteristic that has lead to wide use of conotoxins in ion channel research and as therapeutic agents.

The fact that conotoxins generally contain many disulfide-bonds make them hard to produce – when synthesized as linear peptides, refolding often results in misfolding because cysteines do not form native disulfide-bonds. Recently we have found that cone snails have evolved a hypervariable family of Protein Disulfide Isomerases (PDIs) – enzymes that specifically assist the formation of disulfide-bonds in conotoxins (Safavi-Hemami, H. et al. (2016) Rapid Expansion of the Protein Disulfide Isomerase Gene Family Facilitates the Folding of Venom Peptides. Proc. Natl. Acad. Sci. USA, 113, 3227-3232). The results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails.

We are now in the process of establishing an E. coli expression system that will allow the production and molecular characterization of a variety of conotoxins and other disulfide-rich peptides of biomedical or research interest. The system is based on co-expression with the conotoxin-specific PDIs, and we have shown that we can thus produce conotoxins that have otherwise been impossible to make. Soon, we are planning to extend our work to expression of conotoxins to mammalian cells as well. Moreover, we are determining the biological activity of purified conotoxins in collaboration with a group at the University of Utah.

The project involves a wide variety of techniques in protein biochemistry involving protein expression and purification with potential to extend to cell biological work in mammalian cells. Moreover, there will be possibility to determine NMR structures of conotoxins.
Methods used:A wide variety of protein biochemical methods
Keywords:Protein folding, Conotoxins, Disulfide-bond formation
Supervisor(s): Lars Ellgaard
Email:lellgaard@bio.ku.dk